Neoadjuvant PARPi or chemotherapy in ovarian cancer informs targeting effector Treg cells for homologous-recombination-deficient tumors.

Homologous recombination deficiency (HRD) is prevalent in cancer, sensitizing tumor cells to poly (ADP-ribose) polymerase (PARP) inhibition. However, the impact of HRD and related therapies on the tumor microenvironment (TME) remains elusive. Our study generates single-cell gene expression and T cell receptor profiles, along with validatory multimodal datasets from >100 high-grade serous ovarian cancer (HGSOC) samples, primarily from a phase II clinical trial (NCT04507841). Neoadjuvant monotherapy with the PARP inhibitor (PARPi) niraparib achieves impressive 62.5% and 73.6% response rates per RECIST v.1.1 and GCIG CA125, respectively. We identify effector regulatory T cells (eTregs) as key responders to HRD and neoadjuvant therapies, co-occurring with other tumor-reactive T cells, particularly terminally exhausted CD8+ T cells (Tex). TME-wide interferon signaling correlates with cancer cells upregulating MHC class II and co-inhibitory ligands, potentially driving Treg and Tex fates. Depleting eTregs in HRD mouse models, with or without PARP inhibition, significantly suppresses tumor growth without observable toxicities, underscoring the potential of eTreg-focused therapeutics for HGSOC and other HRD-related tumors.

HIV-1 remission and possible cure in a woman after haplo-cord blood transplant.

Previously, two men were cured of HIV-1 through CCR5Δ32 homozygous (CCR5Δ32/Δ32) allogeneic adult stem cell transplant. We report the first remission and possible HIV-1 cure in a mixed-race woman who received a CCR5Δ32/Δ32 haplo-cord transplant (cord blood cells combined with haploidentical stem cells from an adult) to treat acute myeloid leukemia (AML). Peripheral blood chimerism was 100% CCR5Δ32/Δ32 cord blood by week 14 post-transplant and persisted through 4.8 years of follow-up. Immune reconstitution was associated with (1) loss of detectable replication-competent HIV-1 reservoirs, (2) loss of HIV-1-specific immune responses, (3) in vitro resistance to X4 and R5 laboratory variants, including pre-transplant autologous latent reservoir isolates, and (4) 18 months of HIV-1 control with aviremia, off antiretroviral therapy, starting at 37 months post-transplant. CCR5Δ32/Δ32 haplo-cord transplant achieved remission and a possible HIV-1 cure for a person of diverse ancestry, living with HIV-1, who required a stem cell transplant for acute leukemia.

Mechanisms of readthrough mitigation reveal principles of GCN1-mediated translational quality control.

Readthrough into the 3' untranslated region (3' UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but the underlying mechanisms remain unknown. Here, we show in Caenorhabditis elegans and mammalian cells that readthrough proteins are targeted by a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome-collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions (CTEs) are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay initiated by GCN1 and CCR4/NOT limits the accumulation of readthrough products. Unexpectedly, selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes collide at nonoptimal codons, enriched in 3' UTRs, transmembrane proteins, and collagens. GCN1 dysfunction increasingly perturbs these protein classes during aging, resulting in mRNA and proteome imbalance. Our results define GCN1 as a key factor acting during translation in maintaining protein homeostasis.

Antigen dominance hierarchies shape TCF1+ progenitor CD8 T cell phenotypes in tumors.

CD8 T cell responses against different tumor neoantigens occur simultaneously, yet little is known about the interplay between responses and its impact on T cell function and tumor control. In mouse lung adenocarcinoma, we found that immunodominance is established in tumors, wherein CD8 T cell expansion is predominantly driven by the antigen that most stably binds MHC. T cells responding to subdominant antigens were enriched for a TCF1+ progenitor phenotype correlated with response to immune checkpoint blockade (ICB) therapy. However, the subdominant T cell response did not preferentially benefit from ICB due to a dysfunctional subset of TCF1+ cells marked by CCR6 and Tc17 differentiation. Analysis of human samples and sequencing datasets revealed that CCR6+ TCF1+ cells exist across human cancers and are not correlated with ICB response. Vaccination eliminated CCR6+ TCF1+ cells and dramatically improved the subdominant response, highlighting a strategy to optimally engage concurrent neoantigen responses against tumors.

CXCR6 positions cytotoxic T cells to receive critical survival signals in the tumor microenvironment.

Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses.

Molecular Basis for poly(A) RNP Architecture and Recognition by the Pan2-Pan3 Deadenylase.

The stability of eukaryotic mRNAs is dependent on a ribonucleoprotein (RNP) complex of poly(A)-binding proteins (PABPC1/Pab1) organized on the poly(A) tail. This poly(A) RNP not only protects mRNAs from premature degradation but also stimulates the Pan2-Pan3 deadenylase complex to catalyze the first step of poly(A) tail shortening. We reconstituted this process in vitro using recombinant proteins and show that Pan2-Pan3 associates with and degrades poly(A) RNPs containing two or more Pab1 molecules. The cryo-EM structure of Pan2-Pan3 in complex with a poly(A) RNP composed of 90 adenosines and three Pab1 protomers shows how the oligomerization interfaces of Pab1 are recognized by conserved features of the deadenylase and thread the poly(A) RNA substrate into the nuclease active site. The structure reveals the basis for the periodic repeating architecture at the 3' end of cytoplasmic mRNAs. This illustrates mechanistically how RNA-bound Pab1 oligomers act as rulers for poly(A) tail length over the mRNAs' lifetime.

Structural Basis for Allosteric Ligand Recognition in the Human CC Chemokine Receptor 7.

The CC chemokine receptor 7 (CCR7) balances immunity and tolerance by homeostatic trafficking of immune cells. In cancer, CCR7-mediated trafficking leads to lymph node metastasis, suggesting the receptor as a promising therapeutic target. Here, we present the crystal structure of human CCR7 fused to the protein Sialidase NanA by using data up to 2.1 Å resolution. The structure shows the ligand Cmp2105 bound to an intracellular allosteric binding pocket. A sulfonamide group, characteristic for various chemokine receptor ligands, binds to a patch of conserved residues in the Gi protein binding region between transmembrane helix 7 and helix 8. We demonstrate how structural data can be used in combination with a compound repository and automated thermal stability screening to identify and modulate allosteric chemokine receptor antagonists. We detect both novel (CS-1 and CS-2) and clinically relevant (CXCR1-CXCR2 phase-II antagonist Navarixin) CCR7 modulators with implications for multi-target strategies against cancer.

Allogeneic immunity clears latent virus following allogeneic stem cell transplantation in SIV-infected ART-suppressed macaques.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) from donors lacking C-C chemokine receptor 5 (CCR5Δ32/Δ32) can cure HIV, yet mechanisms remain speculative. To define how alloHSCT mediates HIV cure, we performed MHC-matched alloHSCT in SIV+, anti-retroviral therapy (ART)-suppressed Mauritian cynomolgus macaques (MCMs) and demonstrated that allogeneic immunity was the major driver of reservoir clearance, occurring first in peripheral blood, then peripheral lymph nodes, and finally in mesenteric lymph nodes draining the gastrointestinal tract. While allogeneic immunity could extirpate the latent viral reservoir and did so in two alloHSCT-recipient MCMs that remained aviremic >2.5 years after stopping ART, in other cases, it was insufficient without protection of engrafting cells afforded by CCR5-deficiency, as CCR5-tropic virus spread to donor CD4+ T cells despite full ART suppression. These data demonstrate the individual contributions of allogeneic immunity and CCR5 deficiency to HIV cure and support defining targets of alloimmunity for curative strategies independent of HSCT.

Single-cell atlas of healthy human blood unveils age-related loss of NKG2C+GZMB-CD8+ memory T cells and accumulation of type 2 memory T cells.

Extensive, large-scale single-cell profiling of healthy human blood at different ages is one of the critical pending tasks required to establish a framework for the systematic understanding of human aging. Here, using single-cell RNA/T cell receptor (TCR)/BCR-seq with protein feature barcoding, we profiled 317 samples from 166 healthy individuals aged 25-85 years old. From this, we generated a dataset from ∼2 million cells that described 55 subpopulations of blood immune cells. Twelve subpopulations changed with age, including the accumulation of GZMK+CD8+ T cells and HLA-DR+CD4+ T cells. In contrast to other T cell memory subsets, transcriptionally distinct NKG2C+GZMB-CD8+ T cells counterintuitively decreased with age. Furthermore, we found a concerted age-associated increase in type 2/interleukin (IL)4-expressing memory subpopulations across CD4+ and CD8+ T cell compartments (CCR4+CD8+ Tcm and Th2 CD4+ Tmem), suggesting a systematic functional shift in immune homeostasis with age. Our work provides novel insights into healthy human aging and a comprehensive annotated resource.

A key GPCR phosphorylation motif discovered in arrestin2⋠CCR5 phosphopeptide complexes.

The two non-visual arrestins, arrestin2 and arrestin3, bind hundreds of GPCRs with different phosphorylation patterns, leading to distinct functional outcomes. Structural information on these interactions is available only for very few GPCRs. Here, we have characterized the interactions between the phosphorylated human CC chemokine receptor 5 (CCR5) and arrestin2. We identified several new CCR5 phosphorylation sites necessary for stable arrestin2 complex formation. Structures of arrestin2 in the apo form and complexes with CCR5 C-terminal phosphopeptides, together with NMR, biochemical, and functional assays, revealed three phosphoresidues in a pXpp motif that are essential for arrestin2 binding and activation. The identified motif appears responsible for robust arrestin2 recruitment in many other GPCRs. An analysis of receptor sequences and available structural and functional information provides hints on the molecular basis of arrestin2/arrestin3 isoform specificity. Our findings demonstrate how multi-site phosphorylation controls GPCR⋅arrestin interactions and provide a framework to probe the intricate details of arrestin signaling.

Mechanism of ribosome-associated mRNA degradation during tubulin autoregulation.

Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics and microtubule function. When cells sense excess free tubulin, they trigger degradation of the encoding mRNAs, which requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates the decay of tubulin mRNAs is unknown. Here, our biochemical and structural analysis reveals that TTC5 recruits the poorly studied protein SCAPER to the ribosome. SCAPER, in turn, engages the CCR4-NOT deadenylase complex through its CNOT11 subunit to trigger tubulin mRNA decay. SCAPER mutants that cause intellectual disability and retinitis pigmentosa in humans are impaired in CCR4-NOT recruitment, tubulin mRNA degradation, and microtubule-dependent chromosome segregation. Our findings demonstrate how recognition of a nascent polypeptide on the ribosome is physically linked to mRNA decay factors via a relay of protein-protein interactions, providing a paradigm for specificity in cytoplasmic gene regulation.

Resident cardiac macrophages mediate adaptive myocardial remodeling.

Cardiac macrophages represent a heterogeneous cell population with distinct origins, dynamics, and functions. Recent studies have revealed that C-C Chemokine Receptor 2 positive (CCR2+) macrophages derived from infiltrating monocytes regulate myocardial inflammation and heart failure pathogenesis. Comparatively little is known about the functions of tissue resident (CCR2-) macrophages. Herein, we identified an essential role for CCR2- macrophages in the chronically failing heart. Depletion of CCR2- macrophages in mice with dilated cardiomyopathy accelerated mortality and impaired ventricular remodeling and coronary angiogenesis, adaptive changes necessary to maintain cardiac output in the setting of reduced cardiac contractility. Mechanistically, CCR2- macrophages interacted with neighboring cardiomyocytes via focal adhesion complexes and were activated in response to mechanical stretch through a transient receptor potential vanilloid 4 (TRPV4)-dependent pathway that controlled growth factor expression. These findings establish a role for tissue-resident macrophages in adaptive cardiac remodeling and implicate mechanical sensing in cardiac macrophage activation.

CCR7 Chemokine Receptor-Inducible lnc-Dpf3 Restrains Dendritic Cell Migration by Inhibiting HIF-1α-Mediated Glycolysis.

CCR7 chemokine receptor stimulation induces rapid but transient dendritic cell (DC) migration toward draining lymph nodes, which is critical for the initiation of protective immunity and maintenance of immune homeostasis. The mechanisms for terminating CCR7-mediated DC migration remain incompletely understood. Here we have identified a long non-coding RNA lnc-Dpf3 whose feedback restrained CCR7-mediated DC migration. CCR7 stimulation upregulated lnc-Dpf3 via removing N6-methyladenosine (m6A) modification to prevent RNA degradation. DC-specific lnc-Dpf3 deficiency increased CCR7-mediated DC migration, leading to exaggerated adaptive immune responses and inflammatory injuries. Mechanistically, CCR7 stimulation activated the HIF-1α transcription factor pathway in DCs, leading to metabolic reprogramming toward glycolysis for DC migration. lnc-Dpf3 directly bound to HIF-1α and suppressed HIF-1α-dependent transcription of the glycolytic gene Ldha, thus inhibiting DC glycolytic metabolism and migratory capacity. We demonstrate a critical role for CCR7-inducible lnc-Dpf3 in coupling epigenetic and metabolic pathways to feedback-control DC migration and inflammatory responses.

Metabolite-Sensing Receptor Ffar2 Regulates Colonic Group 3 Innate Lymphoid Cells and Gut Immunity.

Group 3 innate lymphoid cells (ILC3s) sense environmental signals that are critical for gut homeostasis and host defense. However, the metabolite-sensing G-protein-coupled receptors that regulate colonic ILC3s remain poorly understood. We found that colonic ILC3s expressed Ffar2, a microbial metabolite-sensing receptor, and that Ffar2 agonism promoted ILC3 expansion and function. Deficiency of Ffar2 in ILC3s decreased their in situ proliferation and ILC3-derived interleukin-22 (IL-22) production. This led to impaired gut epithelial function characterized by altered mucus-associated proteins and antimicrobial peptides and increased susceptibility to colonic injury and bacterial infection. Ffar2 increased IL-22+ CCR6+ ILC3s and influenced ILC3 abundance in colonic lymphoid tissues. Ffar2 agonism differentially activated AKT or ERK signaling and increased ILC3-derived IL-22 via an AKT and STAT3 axis. Our findings suggest that Ffar2 regulates colonic ILC3 proliferation and function, and they identify an ILC3-receptor signaling pathway modulating gut homeostasis and pathogen defense.

Transcription Dynamics Regulate Poly(A) Tails and Expression of the RNA Degradation Machinery to Balance mRNA Levels.

Gene expression is regulated by the rates of synthesis and degradation of mRNAs, but how these processes are coordinated is poorly understood. Here, we show that reduced transcription dynamics of specific genes leads to enhanced m6A deposition, preferential activity of the CCR4-Not complex, shortened poly(A) tails, and reduced stability of the respective mRNAs. These effects are also exerted by internal ribosome entry site (IRES) elements, which we found to be transcriptional pause sites. However, when transcription dynamics, and subsequently poly(A) tails, are globally altered, cells buffer mRNA levels by adjusting the expression of mRNA degradation machinery. Stress-provoked global impediment of transcription elongation leads to a dramatic inhibition of the mRNA degradation machinery and massive mRNA stabilization. Accordingly, globally enhanced transcription, such as following B cell activation or glucose stimulation, has the opposite effects. This study uncovers two molecular pathways that maintain balanced gene expression in mammalian cells by linking transcription to mRNA stability.

A Quantitative Genetic Interaction Map of HIV Infection.

We have developed a platform for quantitative genetic interaction mapping using viral infectivity as a functional readout and constructed a viral host-dependency epistasis map (vE-MAP) of 356 human genes linked to HIV function, comprising >63,000 pairwise genetic perturbations. The vE-MAP provides an expansive view of the genetic dependencies underlying HIV infection and can be used to identify drug targets and study viral mutations. We found that the RNA deadenylase complex, CNOT, is a central player in the vE-MAP and show that knockout of CNOT1, 10, and 11 suppressed HIV infection in primary T cells by upregulating innate immunity pathways. This phenotype was rescued by deletion of IRF7, a transcription factor regulating interferon-stimulated genes, revealing a previously unrecognized host signaling pathway involved in HIV infection. The vE-MAP represents a generic platform that can be used to study the global effects of how different pathogens hijack and rewire the host during infection.

B-1 lymphocytes in adipose tissue as innate modulators of inflammation linked to cardiometabolic disease.

Fat is stored in distinct depots with unique features in both mice and humans and B cells reside in all adipose depots. We have shown that B cells modulate cardiometabolic disease through activities in two of these key adipose depots: visceral adipose tissue (VAT) and perivascular adipose tissue (PVAT). VAT refers to the adipose tissue surrounding organs, within the abdomen and thorax, and is comprised predominantly of white adipocytes. This depot has been implicated in mediating obesity-related dysmetabolism. PVAT refers to adipose tissue surrounding major arteries. It had long been thought to exist to provide protection and insulation for the vessel, yet recent work demonstrates an important role for PVAT in harboring immune cells, promoting their function and regulating the biology of the underlying vessel. The role of B-2 cells and adaptive immunity in adipose tissue biology has been nicely reviewed elsewhere. Given that, the predominance of B-1 cells in adipose tissue at homeostasis, and the emerging role of B-1 cells in a variety of disease states, we will focus this review on how B-1 cells function in VAT and PVAT depots to promote homeostasis and limit inflammation linked to cardiometabolic disease and factors that regulate this function.

The Chemokine Receptor CCR8 Promotes the Migration of Dendritic Cells into the Lymph Node Parenchyma to Initiate the Allergic Immune Response.

The migration of mature dendritic cells (DCs) into the draining lymph node (dLN) is thought to depend solely on the chemokine receptor CCR7. CD301b+ DCs migrate into the dLN after cutaneous allergen exposure and are required for T helper 2 (Th2) differentiation. We found that CD301b+ DCs poorly upregulated CCR7 expression after allergen exposure and required a second chemokine signal, mediated by CCR8 on CD301b+ DCs and its ligand CCL8, to exit the subcapsular sinus (SCS) and enter the lymph node (LN) parenchyma. After allergen exposure, CD169+SIGN-R1+ macrophages in interfollicular regions produced CCL8, which synergized with CCL21 in a Src-kinase-dependent manner to promote CD301b+ DC migration. In CCR8-deficient mice, CD301b+ DCs remained in the SCS and were unable to enter the LN parenchyma, resulting in defective Th2 differentiation. We have defined a CCR8-dependent stepwise mechanism of DC-subset-specific migration through which LN CD169+SIGN-R1+ macrophages control the polarization of the adaptive immune response.

Endoribonucleolytic Cleavage of m6A-Containing RNAs by RNase P/MRP Complex.

N6-methyladenosine (m6A) is the most abundant internal modification in RNAs and plays regulatory roles in a variety of biological and physiological processes. Despite its important roles, the molecular mechanism underlying m6A-mediated gene regulation is poorly understood. Here, we show that m6A-containing RNAs are subject to endoribonucleolytic cleavage via YTHDF2 (m6A reader protein), HRSP12 (adaptor protein), and RNase P/MRP (endoribonucleases). We demonstrate that HRSP12 functions as an adaptor to bridge YTHDF2 and RNase P/MRP, eliciting rapid degradation of YTHDF2-bound RNAs. Transcriptome-wide analyses show that m6A RNAs that are preferentially targeted for endoribonucleolytic cleavage have an HRSP12-binding site and a RNase P/MRP-directed cleavage site upstream and downstream of the YTHDF2-binding site, respectively. We also find that a subset of m6A-containing circular RNAs associates with YTHDF2 in an HRSP12-dependent manner and is selectively downregulated by RNase P/MRP. Thus, our data expand the known functions of RNase P/MRP to endoribonucleolytic cleavage of m6A RNAs.

Legacy of a magic gene-CCR5-∆32: From discovery to clinical benefit in a generation.

The discovery of the 32-bp deletion allele of the chemokine receptor gene CCR5 showed that homozygous carriers display near-complete resistance to HIV infection, irrespective of exposure. Algorithms of molecular evolutionary theory suggested that the CCR5-∆32 mutation occurred but once in the last millennium and rose by strong selective pressure relatively recently to a ~10% allele frequency in Europeans. Several lines of evidence support the hypothesis that CCR5-∆32 was selected due to its protective influence to resist Yersinia pestis, the agent of the Black Death/bubonic plague of the 14th century. Powerful anti-AIDS entry inhibitors targeting CCR5 were developed as a treatment for HIV patients, particularly those whose systems had developed resistance to powerful anti-retroviral therapies. Homozygous CCR5-∆32/∆32 stem cell transplant donors were used to produce HIV-cleared AIDS patients in at least five "cures" of HIV infection. CCR5 has also been implicated in regulating infection with Staphylococcus aureus, in recovery from stroke, and in ablation of the fatal graft versus host disease (GVHD) in cancer transplant patients. While homozygous CCR5-∆32/32 carriers block HIV infection, alternatively they display an increased risk for encephalomyelitis and death when infected with the West Nile virus.