Bipolar androgen therapy plus nivolumab for patients with metastatic castration-resistant prostate cancer: the COMBAT phase II trial.

Cyclic high-dose testosterone administration, known as bipolar androgen therapy (BAT), is a treatment strategy for patients with metastatic castration-resistant prostate cancer (mCRPC). Here, we report the results of a multicenter, single arm Phase 2 study (NCT03554317) enrolling 45 patients with heavily pretreated mCRPC who received BAT (testosterone cypionate, 400 mg intramuscularly every 28 days) with the addition of nivolumab (480 mg intravenously every 28 days) following three cycles of BAT monotherapy. The primary endpoint of a confirmed PSA50 response rate was met and estimated at 40% (N = 18/45, 95% CI: 25.7-55.7%, P = 0.02 one-sided against the 25% null hypothesis). Sixteen of the PSA50 responses were achieved before the addition of nivolumab. Secondary endpoints included objective response rate (ORR), median PSA progression-free survival, radiographic progression-free survival (rPFS), overall survival (OS), and safety/tolerability. The ORR was 24% (N = 10/42). Three of the objective responses occurred following the addition of nivolumab. After a median follow-up of 17.9 months, the median rPFS was 5.6 (95% CI: 5.4-6.8) months, and median OS was 24.4 (95% CI: 17.6-31.1) months. BAT/nivolumab was well tolerated, resulting in only five (11%) drug related, grade-3 adverse events. In a predefined exploratory analysis, clinical response rates correlated with increased baseline levels of intratumoral PD-1 + T cells. In paired metastatic tumor biopsies, BAT induced pro-inflammatory gene expression changes that were restricted to patients achieving a clinical response. These data suggest that BAT may augment antitumor immune responses that are further potentiated by immune checkpoint blockade.

Prostaglandin E2 controls the metabolic adaptation of T cells to the intestinal microenvironment.

Immune cells must adapt to different environments during the course of an immune response. Here we study the adaptation of CD8+ T cells to the intestinal microenvironment and how this process shapes the establishment of the CD8+ T cell pool. CD8+ T cells progressively remodel their transcriptome and surface phenotype as they enter the gut wall, and downregulate expression of mitochondrial genes. Human and mouse intestinal CD8+ T cells have reduced mitochondrial mass, but maintain a viable energy balance to sustain their function. We find that the intestinal microenvironment is rich in prostaglandin E2 (PGE2), which drives mitochondrial depolarization in CD8+ T cells. Consequently, these cells engage autophagy to clear depolarized mitochondria, and enhance glutathione synthesis to scavenge reactive oxygen species (ROS) that result from mitochondrial depolarization. Impairing PGE2 sensing promotes CD8+ T cell accumulation in the gut, while tampering with autophagy and glutathione negatively impacts the T cell pool. Thus, a PGE2-autophagy-glutathione axis defines the metabolic adaptation of CD8+ T cells to the intestinal microenvironment, to ultimately influence the T cell pool.

FADD- and RIPK3-Mediated Cell Death Ensures Clearance of Ly6Chigh Wound Macrophages from Damaged Tissue.

Cells of the monocyte/macrophage lineage are an integral component of the body's innate ability to restore tissue function after injury. In parallel to mounting an inflammatory response, clearance of monocytes/macrophages from the wound site is critical to re-establish tissue functionality and integrity during the course of healing. The role of regulated cell death in macrophage clearance from damaged tissue and its implications for the outcome of the healing response is little understood. In this study, we explored the role of macrophage-specific FADD-mediated cell death on Ripk3-/- background in a mechanical skin injury model in mice. We found that combined inhibition of RIPK3-mediated necroptosis and FADD-caspase-8-mediated apoptosis in macrophages profoundly delayed wound healing. Importantly, RIPK3 deficiency alone did not considerably alter the wound healing process and macrophage population dynamics, arguing that inhibition of FADD-caspase-8-dependent death of macrophages is primarily responsible for delayed wound closure. Notably, TNF blockade reversed the accumulation of Ly6Chigh macrophages induced by combined deficiency of FADD and RIPK3, indicating a critical dual role of TNF-mediated prosurvival and cell death signaling, particularly in this highly proinflammatory macrophage subset. Our findings reveal a previously uncharacterized cross-talk of inflammatory and cell death signaling in macrophages in regulating repair processes in the skin.

Metabolic and functional remodeling of colonic macrophages in response to high-fat diet-induced obesity.

Little is known about the effects of high-fat diet (HFD)-induced obesity on resident colonic lamina propria (LP) macrophages (LPMs) function and metabolism. Here, we report that obesity and diabetes resulted in increased macrophage infiltration in the colon. These macrophages exhibited the residency phenotype CX3CR1hiMHCIIhi and were CD4-TIM4-. During HFD, resident colonic LPM exhibited a lipid metabolism gene expression signature that overlapped that used to define lipid-associated macrophages (LAMs). Via single-cell RNA sequencing, we identified a sub-cluster of macrophages, increased in HFD, that were responsible for the LAM signature. Compared to other macrophages in the colon, these cells were characterized by elevated glycolysis, phagocytosis, and efferocytosis signatures. CX3CR1hiMHCIIhi colonic resident LPMs had fewer lipid droplets (LDs) and decreased triacylglycerol (TG) content compared to equivalent cells in lean mice and exhibited increased phagocytic capacity, suggesting that HFD induces adaptive responses in LPMs to limit bacterial translocation.

Prostaglandin E 2 controls the metabolic adaptation of T cells to the intestinal microenvironment.

Immune cells must adapt to different environments during the course of an immune response. We studied the adaptation of CD8 + T cells to the intestinal microenvironment and how this process shapes their residency in the gut. CD8 + T cells progressively remodel their transcriptome and surface phenotype as they acquire gut residency, and downregulate expression of mitochondrial genes. Human and mouse gut-resident CD8 + T cells have reduced mitochondrial mass, but maintain a viable energy balance to sustain their function. We found that the intestinal microenvironment is rich in prostaglandin E 2 (PGE 2 ), which drives mitochondrial depolarization in CD8 + T cells. Consequently, these cells engage autophagy to clear depolarized mitochondria, and enhance glutathione synthesis to scavenge reactive oxygen species (ROS) that result from mitochondrial depolarization. Impairing PGE 2 sensing promotes CD8 + T cell accumulation in the gut, while tampering with autophagy and glutathione negatively impacts the T cell population. Thus, a PGE 2 -autophagy-glutathione axis defines the metabolic adaptation of CD8 + T cells to the intestinal microenvironment, to ultimately influence the T cell pool.

Phosphoinositide acyl chain saturation drives CD8+ effector T cell signaling and function.

How lipidome changes support CD8+ effector T (Teff) cell differentiation is not well understood. Here we show that, although naive T cells are rich in polyunsaturated phosphoinositides (PIPn with 3-4 double bonds), Teff cells have unique PIPn marked by saturated fatty acyl chains (0-2 double bonds). PIPn are precursors for second messengers. Polyunsaturated phosphatidylinositol bisphosphate (PIP2) exclusively supported signaling immediately upon T cell antigen receptor activation. In late Teff cells, activity of phospholipase C-γ1, the enzyme that cleaves PIP2 into downstream mediators, waned, and saturated PIPn became essential for sustained signaling. Saturated PIP was more rapidly converted to PIP2 with subsequent recruitment of phospholipase C-γ1, and loss of saturated PIPn impaired Teff cell fitness and function, even in cells with abundant polyunsaturated PIPn. Glucose was the substrate for de novo PIPn synthesis, and was rapidly utilized for saturated PIP2 generation. Thus, separate PIPn pools with distinct acyl chain compositions and metabolic dependencies drive important signaling events to initiate and then sustain effector function during CD8+ T cell differentiation.

Targeting cancer glycosylation repolarizes tumor-associated macrophages allowing effective immune checkpoint blockade.

Immune checkpoint blockade (ICB) has substantially improved the prognosis of patients with cancer, but the majority experiences limited benefit, supporting the need for new therapeutic approaches. Up-regulation of sialic acid-containing glycans, termed hypersialylation, is a common feature of cancer-associated glycosylation, driving disease progression and immune escape through the engagement of Siglec receptors on tumor-infiltrating immune cells. Here, we show that tumor sialylation correlates with distinct immune states and reduced survival in human cancers. The targeted removal of Siglec ligands in the tumor microenvironment, using an antibody-sialidase conjugate, enhanced antitumor immunity and halted tumor progression in several murine models. Using single-cell RNA sequencing, we revealed that desialylation repolarized tumor-associated macrophages (TAMs). We also identified Siglec-E as the main receptor for hypersialylation on TAMs. Last, we found that genetic and therapeutic desialylation, as well as loss of Siglec-E, enhanced the efficacy of ICB. Thus, therapeutic desialylation represents an immunotherapeutic approach to reshape macrophage phenotypes and augment the adaptive antitumor immune response.

Resident TH2 cells orchestrate adipose tissue remodeling at a site adjacent to infection.

Type 2 immunity is associated with adipose tissue (AT) homeostasis and infection with parasitic helminths, but whether AT participates in immunity to these parasites is unknown. We found that the fat content of mesenteric AT (mAT) declined in mice during infection with a gut-restricted helminth. This was associated with the accumulation of metabolically activated, interleukin-33 (IL-33), thymic stromal lymphopoietin (TSLP), and extracellular matrix (ECM)-producing stromal cells. These cells shared transcriptional features, including the expression of Dpp4 and Pi16, with multipotent progenitor cells (MPC) that have been identified in numerous tissues and are reported to be capable of differentiating into fibroblasts and adipocytes. Concomitantly, mAT became infiltrated with resident T helper 2 (TH2) cells that responded to TSLP and IL-33 by producing stromal cell-stimulating cytokines, including transforming growth factor ß1 (TGFß1) and amphiregulin. These TH2 cells expressed genes previously associated with type 2 innate lymphoid cells (ILC2), including Nmur1, Calca, Klrg1, and Arg1, and persisted in mAT for at least 11 months after anthelmintic drug-mediated clearance of infection. We found that MPC and TH2 cells localized to ECM-rich interstitial spaces that appeared shared between mesenteric lymph node, mAT, and intestine. Stromal cell expression of epidermal growth factor receptor (EGFR), the receptor for amphiregulin, was required for immunity to infection. Our findings point to the importance of MPC and TH2 cell interactions within the interstitium in orchestrating AT remodeling and immunity to an intestinal infection.

Androgen receptor activity in prostate cancer dictates efficacy of bipolar androgen therapy through MYC.

Testosterone is the canonical growth factor of prostate cancer but can paradoxically suppress its growth when present at supraphysiological levels. We have previously demonstrated that the cyclical administration of supraphysiological androgen (SPA), termed bipolar androgen therapy (BAT), can result in tumor regression and clinical benefit for patients with castration-resistant prostate cancer. However, predictors and mechanisms of response and resistance have been ill defined. Here, we show that growth inhibition of prostate cancer models by SPA required high androgen receptor (AR) activity and were driven in part by downregulation of MYC. Using matched sequential patient biopsies, we show that high pretreatment AR activity predicted downregulation of MYC, improved clinical response, and prolonged progression-free and overall survival for patients on BAT. BAT induced strong downregulation of AR in all patients, which is shown to be a primary mechanism of acquired resistance to SPA. Acquired resistance was overcome by alternating SPA with the AR inhibitor enzalutamide, which induced adaptive upregulation of AR and resensitized prostate cancer to SPA. This work identifies high AR activity as a predictive biomarker of response to BAT and supports a treatment paradigm for prostate cancer involving alternating between AR inhibition and activation.

An LKB1-mitochondria axis controls TH17 effector function.

CD4+ T cell differentiation requires metabolic reprogramming to fulfil the bioenergetic demands of proliferation and effector function, and enforce specific transcriptional programmes1-3. Mitochondrial membrane dynamics sustains mitochondrial processes4, including respiration and tricarboxylic acid (TCA) cycle metabolism5, but whether mitochondrial membrane remodelling orchestrates CD4+ T cell differentiation remains unclear. Here we show that unlike other CD4+ T cell subsets, T helper 17 (TH17) cells have fused mitochondria with tight cristae. T cell-specific deletion of optic atrophy 1 (OPA1), which regulates inner mitochondrial membrane fusion and cristae morphology6, revealed that TH17 cells require OPA1 for its control of the TCA cycle, rather than respiration. OPA1 deletion amplifies glutamine oxidation, leading to impaired NADH/NAD+ balance and accumulation of TCA cycle metabolites and 2-hydroxyglutarate-a metabolite that influences the epigenetic landscape5,7. Our multi-omics approach revealed that the serine/threonine kinase liver-associated kinase B1 (LKB1) couples mitochondrial function to cytokine expression in TH17 cells by regulating TCA cycle metabolism and transcriptional remodelling. Mitochondrial membrane disruption activates LKB1, which restrains IL-17 expression. LKB1 deletion restores IL-17 expression in TH17 cells with disrupted mitochondrial membranes, rectifying aberrant TCA cycle glutamine flux, balancing NADH/NAD+ and preventing 2-hydroxyglutarate production from the promiscuous activity of the serine biosynthesis enzyme phosphoglycerate dehydrogenase (PHGDH). These findings identify OPA1 as a major determinant of TH17 cell function, and uncover LKB1 as a sensor linking mitochondrial cues to effector programmes in TH17 cells.

Trained immunity of alveolar macrophages requires metabolic rewiring and type 1 interferon signaling.

Environmental microbial triggers shape the development and functionality of the immune system. Alveolar macrophages (AMs), tissue-resident macrophages of the lungs, are in constant and direct contact with inhaled particles and microbes. Such exposures likely impact AM reactivity to subsequent challenges by immunological imprinting mechanisms referred to as trained immunity. Here, we investigated whether a ubiquitous microbial compound has the potential to induce AM training in vivo. We discovered that intranasal exposure to ambient amounts of lipopolysaccharide (LPS) induced a pronounced AM memory response, characterized by enhanced reactivity upon pneumococcal challenge. Exploring the mechanistic basis of AM training, we identified a critical role of type 1 interferon signaling and found that inhibition of fatty acid oxidation and glutaminolysis significantly attenuated the training effect. Notably, adoptive transfer of trained AMs resulted in increased bacterial loads and tissue damage upon subsequent pneumococcal infection. In contrast, intranasal pre-exposure to LPS promoted bacterial clearance, highlighting the complexity of stimulus-induced immune responses, which likely involve multiple cell types and may depend on the local immunological and metabolic environment. Collectively, our findings demonstrate the profound impact of ambient microbial exposure on pulmonary immune memory and reveal tissue-specific features of trained immunity.

Intracellular infection and immune system cues rewire adipocytes to acquire immune function.

Adipose tissue (AT) plays a central role in systemic metabolic homeostasis, but its function during bacterial infection remains unclear. Following subcutaneous bacterial infection, adipocytes surrounding draining lymph nodes initiated a transcriptional response indicative of stimulation with IFN-γ and a shift away from lipid metabolism toward an immunologic function. Natural killer (NK) and invariant NK T (iNKT) cells were identified as sources of infection-induced IFN-γ in perinodal AT (PAT). IFN-γ induced Nos2 expression in adipocytes through a process dependent on nuclear-binding oligomerization domain 1 (NOD1) sensing of live intracellular bacteria. iNOS expression was coupled to metabolic rewiring, inducing increased diversion of extracellular L-arginine through the arginosuccinate shunt and urea cycle to produce nitric oxide (NO), directly mediating bacterial clearance. In vivo, control of infection in adipocytes was dependent on adipocyte-intrinsic sensing of IFN-γ and expression of iNOS. Thus, adipocytes are licensed by innate lymphocytes to acquire anti-bacterial functions during infection.

A common framework of monocyte-derived macrophage activation.

Macrophages populate every organ during homeostasis and disease, displaying features of tissue imprinting and heterogeneous activation. The disconnected picture of macrophage biology that has emerged from these observations is a barrier for integration across models or with in vitro macrophage activation paradigms. We set out to contextualize macrophage heterogeneity across mouse tissues and inflammatory conditions, specifically aiming to define a common framework of macrophage activation. We built a predictive model with which we mapped the activation of macrophages across 12 tissues and 25 biological conditions, finding a notable commonality and finite number of transcriptional profiles, in particular among infiltrating macrophages, which we modeled as defined stages along four conserved activation paths. These activation paths include a "phagocytic" regulatory path, an "inflammatory" cytokine-producing path, an "oxidative stress" antimicrobial path, or a "remodeling" extracellular matrix deposition path. We verified this model with adoptive cell transfer experiments and identified transient RELMɑ expression as a feature of monocyte-derived macrophage tissue engraftment. We propose that this integrative approach of macrophage classification allows the establishment of a common predictive framework of monocyte-derived macrophage activation in inflammation and homeostasis.

Gene-selective transcription promotes the inhibition of tissue reparative macrophages by TNF.

Anti-TNF therapies are a core anti-inflammatory approach for chronic diseases such as rheumatoid arthritis and Crohn's Disease. Previously, we and others found that TNF blocks the emergence and function of alternative-activated or M2 macrophages involved in wound healing and tissue-reparative functions. Conceivably, anti-TNF drugs could mediate their protective effects in part by an altered balance of macrophage activity. To understand the mechanistic basis of how TNF regulates tissue-reparative macrophages, we used RNAseq, scRNAseq, ATACseq, time-resolved phospho-proteomics, gene-specific approaches, metabolic analysis, and signaling pathway deconvolution. We found that TNF controls tissue-reparative macrophage gene expression in a highly gene-specific way, dependent on JNK signaling via the type 1 TNF receptor on specific populations of alternative-activated macrophages. We further determined that JNK signaling has a profound and broad effect on activated macrophage gene expression. Our findings suggest that TNF's anti-M2 effects evolved to specifically modulate components of tissue and reparative M2 macrophages and TNF is therefore a context-specific modulator of M2 macrophages rather than a pan-M2 inhibitor.

Plasmacytoid dendritic cell activation is dependent on coordinated expression of distinct amino acid transporters.

Human plasmacytoid dendritic cells (pDCs) are interleukin-3 (IL-3)-dependent cells implicated in autoimmunity, but the role of IL-3 in pDC biology is poorly understood. We found that IL-3-induced Janus kinase 2-dependent expression of SLC7A5 and SLC3A2, which comprise the large neutral amino acid transporter, was required for mammalian target of rapamycin complex 1 (mTORC1) nutrient sensor activation in response to toll-like receptor agonists. mTORC1 facilitated increased anabolic activity resulting in type I interferon, tumor necrosis factor, and chemokine production and the expression of the cystine transporter SLC7A11. Loss of function of these amino acid transporters synergistically blocked cytokine production by pDCs. Comparison of in vitro-activated pDCs with those from lupus nephritis lesions identified not only SLC7A5, SLC3A2, and SLC7A11 but also ectonucleotide pyrophosphatase-phosphodiesterase 2 (ENPP2) as components of a shared transcriptional signature, and ENPP2 inhibition also blocked cytokine production. Our data identify additional therapeutic targets for autoimmune diseases in which pDCs are implicated.

Mitochondrial metabolism coordinates stage-specific repair processes in macrophages during wound healing.

Wound healing is a coordinated process that initially relies on pro-inflammatory macrophages, followed by a pro-resolution function of these cells. Changes in cellular metabolism likely dictate these distinct activities, but the nature of these changes has been unclear. Here, we profiled early- versus late-stage skin wound macrophages in mice at both the transcriptional and functional levels. We found that glycolytic metabolism in the early phase is not sufficient to ensure productive repair. Instead, by combining conditional disruption of the electron transport chain with deletion of mitochondrial aspartyl-tRNA synthetase, followed by single-cell sequencing analysis, we found that a subpopulation of early-stage wound macrophages are marked by mitochondrial ROS (mtROS) production and HIF1α stabilization, which ultimately drives a pro-angiogenic program essential for timely healing. In contrast, late-phase, pro-resolving wound macrophages are marked by IL-4Rα-mediated mitochondrial respiration and mitohormesis. Collectively, we identify changes in mitochondrial metabolism as a critical control mechanism for macrophage effector functions during wound healing.

Polyamine metabolism is a central determinant of helper T cell lineage fidelity.

Polyamine synthesis represents one of the most profound metabolic changes during T cell activation, but the biological implications of this are scarcely known. Here, we show that polyamine metabolism is a fundamental process governing the ability of CD4+ helper T cells (TH) to polarize into different functional fates. Deficiency in ornithine decarboxylase, a crucial enzyme for polyamine synthesis, results in a severe failure of CD4+ T cells to adopt correct subset specification, underscored by ectopic expression of multiple cytokines and lineage-defining transcription factors across TH cell subsets. Polyamines control TH differentiation by providing substrates for deoxyhypusine synthase, which synthesizes the amino acid hypusine, and mice in which T cells are deficient for hypusine develop severe intestinal inflammatory disease. Polyamine-hypusine deficiency caused widespread epigenetic remodeling driven by alterations in histone acetylation and a re-wired tricarboxylic acid (TCA) cycle. Thus, polyamine metabolism is critical for maintaining the epigenome to focus TH cell subset fidelity.

Fever supports CD8+ effector T cell responses by promoting mitochondrial translation.

Fever can provide a survival advantage during infection. Metabolic processes are sensitive to environmental conditions, but the effect of fever on T cell metabolism is not well characterized. We show that in activated CD8+ T cells, exposure to febrile temperature (39 °C) augmented metabolic activity and T cell effector functions, despite having a limited effect on proliferation or activation marker expression. Transcriptional profiling revealed an up-regulation of mitochondrial pathways, which was consistent with increased mass and metabolism observed in T cells exposed to 39 °C. Through in vitro and in vivo models, we determined that mitochondrial translation is integral to the enhanced metabolic activity and function of CD8+ T cells exposed to febrile temperature. Transiently exposing donor lymphocytes to 39 °C prior to infusion in a myeloid leukemia mouse model conferred enhanced therapeutic efficacy, raising the possibility that exposure of T cells to febrile temperatures could have clinical potential.

IL-33 expression in response to SARS-CoV-2 correlates with seropositivity in COVID-19 convalescent individuals.

Our understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still developing. We perform an observational study to investigate seroprevalence and immune responses in subjects professionally exposed to SARS-CoV-2 and their family members (155 individuals; ages 5-79 years). Seropositivity for SARS-CoV-2 Spike glycoprotein aligns with PCR results that confirm the previous infection. Anti-Spike IgG/IgM titers remain high 60 days post-infection and do not strongly associate with symptoms, except for fever. We analyze PBMCs from a subset of seropositive and seronegative adults. TLR7 agonist-activation reveals an increased population of IL-6+TNF-IL-1ß+ monocytes, while SARS-CoV-2 peptide stimulation elicits IL-33, IL-6, IFNa2, and IL-23 expression in seropositive individuals. IL-33 correlates with CD4+ T cell activation in PBMCs from convalescent subjects and is likely due to T cell-mediated effects on IL-33-producing cells. IL-33 is associated with pulmonary infection and chronic diseases like asthma and COPD, but its role in COVID-19 is unknown. Analysis of published scRNAseq data of bronchoalveolar lavage fluid (BALF) from patients with mild to severe COVID-19 reveals a population of IL-33-producing cells that increases with the disease. Together these findings show that IL-33 production is linked to SARS-CoV-2 infection and warrant further investigation of IL-33 in COVID-19 pathogenesis and immunity.

Sulfur sequestration promotes multicellularity during nutrient limitation.

The behaviour of Dictyostelium discoideum depends on nutrients1. When sufficient food is present these amoebae exist in a unicellular state, but upon starvation they aggregate into a multicellular organism2,3. This biology makes D. discoideum an ideal model for investigating how fundamental metabolism commands cell differentiation and function. Here we show that reactive oxygen species-generated as a consequence of nutrient limitation-lead to the sequestration of cysteine in the antioxidant glutathione. This sequestration limits the use of the sulfur atom of cysteine in processes that contribute to mitochondrial metabolism and cellular proliferation, such as protein translation and the activity of enzymes that contain an iron-sulfur cluster. The regulated sequestration of sulfur maintains D. discoideum in a nonproliferating state that paves the way for multicellular development. This mechanism of signalling through reactive oxygen species highlights oxygen and sulfur as simple signalling molecules that dictate cell fate in an early eukaryote, with implications for responses to nutrient fluctuations in multicellular eukaryotes.