ABSTRACT
Skin-resident CD8+ T cells include distinct interferon-γ-producing [tissue-resident memory T type 1 (TRM1)] and interleukin-17 (IL-17)-producing (TRM17) subsets that differentially contribute to immune responses. However, whether these populations use common mechanisms to establish tissue residence is unknown. In this work, we show that TRM1 and TRM17 cells navigate divergent trajectories to acquire tissue residency in the skin. TRM1 cells depend on a T-bet-Hobit-IL-15 axis, whereas TRM17 cells develop independently of these factors. Instead, c-Maf commands a tissue-resident program in TRM17 cells parallel to that induced by Hobit in TRM1 cells, with an ICOS-c-Maf-IL-7 axis pivotal to TRM17 cell commitment. Accordingly, by targeting this pathway, skin TRM17 cells can be ablated without compromising their TRM1 counterparts. Thus, skin-resident T cells rely on distinct molecular circuitries, which can be exploited to strategically modulate local immunity.
Subject(s)
CD8-Positive T-Lymphocytes , Immunologic Memory , Memory T Cells , Skin , CD8-Positive T-Lymphocytes/immunology , Memory T Cells/immunology , Skin/immunology , Humans , Th17 Cells/immunology , Inducible T-Cell Co-Stimulator Ligand/metabolism , Proto-Oncogene Proteins c-maf/metabolism , Interleukin-7/metabolismABSTRACT
CD8+ tumor-infiltrating lymphocytes with a tissue-resident memory T (TRM) cell phenotype are associated with favorable prognosis in patients with triple-negative breast cancer (TNBC). However, the relative contribution of CD8+ TRM cells to anti-tumor immunity and immune checkpoint blockade efficacy in breast cancer remains unknown. Here, we show that intratumoral CD8+ T cells in murine mammary tumors transcriptionally resemble those from TNBC patients. Phenotypic and transcriptional studies established two intratumoral sub-populations: one more enriched in markers of terminal exhaustion (TEX-like) and the other with a bona fide resident phenotype (TRM-like). Treatment with anti-PD-1 and anti-CTLA-4 therapy resulted in expansion of these intratumoral populations, with the TRM-like subset displaying significantly enhanced cytotoxic capacity. TRM-like CD8+ T cells could also provide local immune protection against tumor rechallenge and a TRM gene signature extracted from tumor-free tissue was significantly associated with improved clinical outcomes in TNBC patients treated with checkpoint inhibitors.
Subject(s)
CD8-Positive T-Lymphocytes , Triple Negative Breast Neoplasms , Humans , Animals , Mice , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Immunologic Memory , Phenotype , Prognosis , Lymphocytes, Tumor-InfiltratingABSTRACT
Accurate identification and effective removal of unwanted variation is essential to derive meaningful biological results from RNA sequencing (RNA-seq) data, especially when the data come from large and complex studies. Using RNA-seq data from The Cancer Genome Atlas (TCGA), we examined several sources of unwanted variation and demonstrate here how these can significantly compromise various downstream analyses, including cancer subtype identification, association between gene expression and survival outcomes and gene co-expression analysis. We propose a strategy, called pseudo-replicates of pseudo-samples (PRPS), for deploying our recently developed normalization method, called removing unwanted variation III (RUV-III), to remove the variation caused by library size, tumor purity and batch effects in TCGA RNA-seq data. We illustrate the value of our approach by comparing it to the standard TCGA normalizations on several TCGA RNA-seq datasets. RUV-III with PRPS can be used to integrate and normalize other large transcriptomic datasets coming from multiple laboratories or platforms.
Subject(s)
Neoplasms , RNA , Humans , Gene Expression Profiling/methods , Sequence Analysis, RNA , Neoplasms/geneticsABSTRACT
Tissue-resident memory T cells (TRM cells) provide rapid and superior control of localized infections. While the transcription factor Runx3 is a critical regulator of CD8+ T cell tissue residency, its expression is repressed in CD4+ T cells. Here, we show that, as a direct consequence of this Runx3-deficiency, CD4+ TRM cells lacked the transforming growth factor (TGF)-ß-responsive transcriptional network that underpins the tissue residency of epithelial CD8+ TRM cells. While CD4+ TRM cell formation required Runx1, this, along with the modest expression of Runx3 in CD4+ TRM cells, was insufficient to engage the TGF-ß-driven residency program. Ectopic expression of Runx3 in CD4+ T cells incited this TGF-ß-transcriptional network to promote prolonged survival, decreased tissue egress, a microanatomical redistribution towards epithelial layers and enhanced effector functionality. Thus, our results reveal distinct programming of tissue residency in CD8+ and CD4+ TRM cell subsets that is attributable to divergent Runx3 activity.
Subject(s)
Immunologic Memory , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Transforming Growth Factor beta/metabolismABSTRACT
The spleen is a compartmentalized organ that serves as a blood filter and safeguard of systemic immune surveillance. Labyrinthine networks of fibroblastic stromal cells construct complex niches within the white pulp and red pulp that are important for tissue homeostasis and immune activation. However, the identity and roles of the global splenic fibroblastic stromal cells in homeostasis and immune responses are poorly defined. Here, we performed a cellular and molecular dissection of the splenic reticular stromal cell landscape. We found that white pulp fibroblastic reticular cells (FRCs) responded robustly during acute viral infection, but this program of gene regulation was suppressed during persistent viral infection. Single-cell transcriptomic analyses in mice revealed diverse fibroblast cell niches and unexpected heterogeneity among podoplanin-expressing cells that include glial, mesothelial, and adventitial cells in addition to FRCs. We found analogous fibroblastic stromal cell diversity in the human spleen. In addition, we identify the transcription factor SpiB as a critical regulator required to support white pulp FRC differentiation, homeostatic chemokine expression, and antiviral T cell responses. Together, our study provides a comprehensive map of fibroblastic stromal cell types in the spleen and defines roles for red and white pulp fibroblasts for splenic function and orchestration of immune responses.
Subject(s)
Fibroblasts/immunology , Homeostasis/immunology , Spleen/immunology , Stromal Cells/immunology , Animals , Cell Differentiation , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , T-Lymphocytes/immunologyABSTRACT
The autoimmune regulator (AIRE) induces the transcription of thousands of peripheral tissue genes (PTGs) in thymic epithelial cells (TECs) to mediate immunological tolerance. The chromatin state required for optimal AIRE function in TECs and how this state is induced remains unclear. We tested the role of the histone acetyltransferase, KAT7 (also known as HBO1 or MYST2), which is essential for acetylation of histone 3 lysine 14, in TEC differentiation, AIRE-mediated PTG expression, and thymic tolerance. We find that KAT7 is required for optimal expansion of medullary TEC and has a major role in the expression of AIRE-dependent PTGs, associated with enhanced chromatin accessibility at these gene loci in TECs. Mice with TEC-specific Kat7 deletion develop organ-specific autoimmunity with features resembling those observed in Aire-deficient mice. These findings highlight critical roles for KAT7-mediated acetylation in promoting a chromatin state at PTG loci that enables AIRE function and the establishment of immunological tolerance.
Subject(s)
Epithelial Cells/immunology , Histone Acetyltransferases/immunology , Thymus Gland/immunology , Transcription Factors/immunology , Animals , Immune Tolerance/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Thymus Gland/cytology , AIRE ProteinABSTRACT
Tissue-resident memory T (TRM) cells are non-recirculating cells that exist throughout the body. Although TRM cells in various organs rely on common transcriptional networks to establish tissue residency, location-specific factors adapt these cells to their tissue of lodgment. Here we analyze TRM cell heterogeneity between organs and find that the different environments in which these cells differentiate dictate TRM cell function, durability and malleability. We find that unequal responsiveness to TGFß is a major driver of this diversity. Notably, dampened TGFß signaling results in CD103- TRM cells with increased proliferative potential, enhanced function and reduced longevity compared with their TGFß-responsive CD103+ TRM counterparts. Furthermore, whereas CD103- TRM cells readily modified their phenotype upon relocation, CD103+ TRM cells were comparatively resistant to transdifferentiation. Thus, despite common requirements for TRM cell development, tissue adaptation of these cells confers discrete functional properties such that TRM cells exist along a spectrum of differentiation potential that is governed by their local tissue microenvironment.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Cell Plasticity/immunology , Cellular Microenvironment/immunology , Immunologic Memory/immunology , Animals , Antigens, CD/immunology , CD8-Positive T-Lymphocytes/cytology , Female , Integrin alpha Chains/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction/immunology , Transforming Growth Factor beta1/metabolismABSTRACT
Macrophages are diverse immune cells that reside in all tissues. Although macrophages have been implicated in mammary-gland function, their diversity has not been fully addressed. By exploiting high-resolution three-dimensional imaging and flow cytometry, we identified a unique population of tissue-resident ductal macrophages that form a contiguous network between the luminal and basal layers of the epithelial tree throughout postnatal development. Ductal macrophages are long lived and constantly survey the epithelium through dendrite movement, revealed via advanced intravital imaging. Although initially originating from embryonic precursors, ductal macrophages derive from circulating monocytes as they expand during puberty. Moreover, they undergo proliferation in pregnancy to maintain complete coverage of the epithelium in lactation, when they are poised to phagocytose milk-producing cells post-lactation and facilitate remodelling. Interestingly, ductal macrophages strongly resemble mammary tumour macrophages and form a network that pervades the tumour. Thus, the mammary epithelium programs specialized resident macrophages in both physiological and tumorigenic contexts.
Subject(s)
Epithelial Cells/physiology , Epithelium/physiology , Animals , Cell Proliferation/physiology , Female , Lactation/physiology , Macrophages/physiology , Mammary Glands, Animal/physiology , Mice , Mice, Inbred C57BL , Monocytes/physiology , Phagocytosis/physiology , PregnancyABSTRACT
Venetoclax, a potent and selective BCL2 inhibitor, synergizes with endocrine therapy in preclinical models of ER-positive breast cancer. Using a phase Ib 3 + 3 dose-escalation and expansion study design, 33 patients with ER and BCL2-positive metastatic disease (mean prior regimens, 2; range, 0-8) were treated with daily tamoxifen (20 mg) and venetoclax (200-800 mg). Apart from uncomplicated "on-target" lymphopenia, no dose-limiting toxicities or high-grade adverse events were observed in the escalation phase (15 patients), and 800 mg was selected as the recommended phase II dose (RP2D). In the expansion phase (18 patients), few high-grade treatment-related adverse events were observed. For 24 patients treated at the RP2D, the confirmed radiologic response rate was 54% and the clinical benefit rate was 75%. Treatment responses were preempted by metabolic responses (FDG-PET) at 4 weeks and correlated with serial changes in circulating tumor DNA. Radiologic responses (40%) and clinical benefit (70%) were observed in 10 patients with plasma-detected ESR1 mutations. SIGNIFICANCE: In the first clinical study to evaluate venetoclax in a solid tumor, we demonstrate that combining venetoclax with endocrine therapy has a tolerable safety profile and elicits notable activity in ER and BCL2-positive metastatic breast cancer. These findings support further investigation of combination therapy for patients with BCL2-positive tumors.See related commentary by Drago et al., p. 323.This article is highlighted in the In This Issue feature, p. 305.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Estrogen Receptor alpha/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/pharmacokinetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Circulating Tumor DNA/analysis , Disease-Free Survival , Dose-Response Relationship, Drug , Drug Administration Schedule , Estrogen Receptor alpha/metabolism , Female , Humans , Middle Aged , Neoplasm Metastasis , Proto-Oncogene Proteins c-bcl-2/metabolism , Sulfonamides/administration & dosage , Tamoxifen/administration & dosage , Tissue DistributionABSTRACT
Classification on the basis of gene expression data derived from RNA-seq promises to become an important part of modern medicine. We propose a new classification method based on a model where the data is marginally negative binomial but dependent, thereby incorporating the dependence known to be present between measurements from different genes. The method, called qtQDA, works by first performing a quantile transformation (qt) then applying Gaussian quadratic discriminant analysis (QDA) using regularized covariance matrix estimates. We show that qtQDA has excellent performance when applied to real data sets and has advantages over some existing approaches. An R package implementing the method is also available on https://github.com/goknurginer/qtQDA.
ABSTRACT
Unwanted variation can be highly problematic and so its detection is often crucial. Relative log expression (RLE) plots are a powerful tool for visualizing such variation in high dimensional data. We provide a detailed examination of these plots, with the aid of examples and simulation, explaining what they are and what they can reveal. RLE plots are particularly useful for assessing whether a procedure aimed at removing unwanted variation, i.e. a normalization procedure, has been successful. These plots, while originally devised for gene expression data from microarrays, can also be used to reveal unwanted variation in many other kinds of high dimensional data, where such variation can be problematic.
Subject(s)
Computer Simulation , Gene ExpressionABSTRACT
T-cell differentiation is governed by interactions with thymic epithelial cells (TECs) and defects in this process undermine immune function and tolerance. To uncover new strategies to restore thymic function and adaptive immunity in immunodeficiency, we sought to determine the molecular mechanisms that control life and death decisions in TECs. Guided by gene expression profiling, we created mouse models that specifically deleted prosurvival genes in TECs. We found that although BCL-2 and BCL-XL were dispensable for TEC homeostasis, MCL-1 deficiency impacted on TECs as early as embryonic day 15.5, resulting in early thymic atrophy and T-cell lymphopenia, with near complete loss of thymic tissue by 2 months of age. MCL-1 was not necessary for TEC differentiation but was continually required for the survival of mature cortical and medullary TECs and the maintenance of thymic architecture. A screen of TEC trophic factors in organ cultures showed that epidermal growth factor upregulated MCL-1 via MAPK/ERK kinase activity, providing a molecular mechanism for the support of TEC survival. This signaling axis governing TEC survival and thymic function represents a new target for strategies for thymic protection and regeneration.
Subject(s)
Cell Survival/genetics , Epithelial Cells/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Thymus Gland/physiology , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Survival/drug effects , Epidermal Growth Factor/pharmacology , Epithelial Cells/drug effects , Female , Gene Expression , Gene Expression Profiling , Gene Expression Regulation , Gene Knockdown Techniques , Homeostasis/genetics , Immunophenotyping , Lymphopenia/genetics , Male , Mice , Mice, Knockout , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Thymocytes/cytology , Thymocytes/immunology , Thymocytes/metabolism , Thymus Gland/pathology , bcl-X Protein/genetics , bcl-X Protein/metabolismABSTRACT
We present a method for estimating the age of a mutation based on the genetic length of ancestral haplotypes shared between individuals carrying the mutation. The method can be reliably applied to small samples, typical of situations involving rare mutations, and makes effective use of modern high-density SNP data, thus overcoming two of the limitations with existing methods. The method provides age estimates and confidence intervals without the use of asymptotic theory and is applicable to genealogies in which the data are independent or correlated. In the correlated case we estimate the correlation directly from the data, rather than relying on a model for the genealogy. To demonstrate the method's efficacy, we provide simulation results and compare it to other methods. The length data are obtained with a simple procedure, and an R script is available for performing the calculations.
Subject(s)
Evolution, Molecular , Genetic Markers , Mutation , Chromosome Mapping , Computer Simulation , DNA Mutational Analysis , Genetic Loci , Haplotypes , Humans , Linkage Disequilibrium , Models, Genetic , Polymorphism, Single NucleotideABSTRACT
Leigh syndrome (LS) is a severe neurodegenerative disorder with characteristic bilateral lesions, typically in the brainstem and basal ganglia. It usually presents in infancy and is genetically heterogeneous, but most individuals with mitochondrial complex IV (or cytochrome c oxidase) deficiency have mutations in the biogenesis factor SURF1. We studied eight complex IV-deficient LS individuals from six families of Lebanese origin. They differed from individuals with SURF1 mutations in having seizures as a prominent feature. Complementation analysis suggested they had mutation(s) in the same gene but targeted massively parallel sequencing (MPS) of 1,034 genes encoding known mitochondrial proteins failed to identify a likely candidate. Linkage and haplotype analyses mapped the location of the gene to chromosome 19 and targeted MPS of the linkage region identified a homozygous c.3G>C (p.Met1?) mutation in C19orf79. Abolishing the initiation codon could potentially still allow initiation at a downstream methionine residue but we showed that this would not result in a functional protein. We confirmed that mutation of this gene was causative by lentiviral-mediated phenotypic correction. C19orf79 was recently renamed PET100 and predicted to encode a complex IV biogenesis factor. We showed that it is located in the mitochondrial inner membrane and forms a â¼300 kDa subcomplex with complex IV subunits. Previous proteomic analyses of mitochondria had overlooked PET100 because its small size was below the cutoff for annotating bona fide proteins. The mutation was estimated to have arisen at least 520 years ago, explaining how the families could have different religions and different geographic origins within Lebanon.
Subject(s)
Cytochrome-c Oxidase Deficiency/ethnology , Cytochrome-c Oxidase Deficiency/genetics , Founder Effect , Leigh Disease/ethnology , Leigh Disease/genetics , Mitochondrial Proteins/genetics , Chromosomes, Human, Pair 19/genetics , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Cytochrome-c Oxidase Deficiency/complications , DNA, Mitochondrial/genetics , DNA, Mitochondrial/isolation & purification , Female , Genetic Complementation Test , Genetic Linkage , Genome-Wide Association Study , Haplotypes , Homozygote , Humans , Infant , Lebanon , Leigh Disease/complications , Male , Mitochondria/genetics , Mitochondria/metabolism , Mutation , Pedigree , Polymorphism, Single Nucleotide , Proteomics , Sequence Analysis, DNAABSTRACT
We previously identified a homozygous mutation in the Golgi SNAP receptor complex 2 gene (GOSR2) in six patients with progressive myoclonus epilepsy. To define the syndrome better we analysed the clinical and electrophysiological phenotype in 12 patients with GOSR2 mutations, including six new unrelated subjects. Clinical presentation was remarkably similar with early onset ataxia (average 2 years of age), followed by myoclonic seizures at the average age of 6.5 years. Patients developed multiple seizure types, including generalized tonic clonic seizures, absence seizures and drop attacks. All patients developed scoliosis by adolescence, making this an important diagnostic clue. Additional skeletal deformities were present, including pes cavus in four patients and syndactyly in two patients. All patients had elevated serum creatine kinase levels (median 734 IU) in the context of normal muscle biopsies. Electroencephalography revealed pronounced generalized spike and wave discharges with a posterior predominance and photosensitivity in all patients, with focal EEG features seen in seven patients. The disease course showed a relentless decline; patients uniformly became wheelchair bound (mean age 13 years) and four had died during their third or early fourth decade. All 12 cases had the same variant (c.430G>T, G144W) and haplotype analyses confirmed a founder effect. The cases all came from countries bounding the North Sea, extending to the coastal region of Northern Norway. 'North Sea' progressive myoclonus epilepsy has a homogeneous clinical presentation and relentless disease course allowing ready identification from the other progressive myoclonus epilepsies.
Subject(s)
Mutation , Myoclonic Epilepsies, Progressive/genetics , Myoclonic Epilepsies, Progressive/physiopathology , Phenotype , Qb-SNARE Proteins/genetics , Adolescent , Adult , Ataxia/genetics , Ataxia/physiopathology , Child , Electroencephalography , Europe , Female , Humans , Male , Mutation/genetics , Myoclonic Epilepsies, Progressive/mortality , North Sea , Young AdultABSTRACT
Autosomal dominant congenital spinal muscular atrophy is characterized by predominantly lower limb weakness and wasting, and congenital or early-onset contractures of the hip, knee and ankle. Mutations in TRPV4, encoding a cation channel, have recently been identified in one large dominant congenital spinal muscular atrophy kindred, but the genetic basis of dominant congenital spinal muscular atrophy in many families remains unknown. It has been hypothesized that differences in the timing and site of anterior horn cell loss in the central nervous system account for the variations in clinical phenotype between different forms of spinal muscular atrophy, but there has been a lack of neuropathological data to support this concept in dominant congenital spinal muscular atrophy. We report clinical, electrophysiology, muscle magnetic resonance imaging and histopathology findings in a four generation family with typical dominant congenital spinal muscular atrophy features, without mutations in TRPV4, and in whom linkage to other known dominant neuropathy and spinal muscular atrophy genes has been excluded. The autopsy findings in the proband, who died at 14 months of age from an unrelated illness, provided a rare opportunity to study the neuropathological basis of dominant congenital spinal muscular atrophy. There was a reduction in anterior horn cell number in the lumbar and, to a lesser degree, the cervical spinal cord, and atrophy of the ventral nerve roots at these levels, in the absence of additional peripheral nerve pathology or abnormalities elsewhere along the neuraxis. Despite the young age of the child at the time of autopsy, there was no pathological evidence of ongoing loss or degeneration of anterior horn cells suggesting that anterior horn cell loss in dominant congenital spinal muscular atrophy occurs in early life, and is largely complete by the end of infancy. These findings confirm that dominant congenital spinal muscular atrophy is a true form of spinal muscular atrophy caused by a loss of anterior horn cells localized to lumbar and cervical regions early in development.
Subject(s)
Anterior Horn Cells/pathology , Family Health , Genetic Linkage , Muscular Atrophy, Spinal/genetics , Muscular Atrophy, Spinal/pathology , TRPV Cation Channels/genetics , Aged , Autopsy , Child , Child, Preschool , Female , Humans , Infant , Magnetic Resonance Imaging , Male , Muscle, Skeletal/pathology , Muscle, Skeletal/physiopathology , Muscular Atrophy, Spinal/complications , Myosins/metabolism , Phenotype , Ultrasonography, DopplerABSTRACT
The progressive myoclonus epilepsies (PMEs) are a group of predominantly recessive disorders that present with action myoclonus, tonic-clonic seizures, and progressive neurological decline. Many PMEs have similar clinical presentations yet are genetically heterogeneous, making accurate diagnosis difficult. A locus for PME was mapped in a consanguineous family with a single affected individual to chromosome 17q21. An identical-by-descent, homozygous mutation in GOSR2 (c.430G>T, p.Gly144Trp), a Golgi vesicle transport gene, was identified in this patient and in four apparently unrelated individuals. A comparison of the phenotypes in these patients defined a clinically distinct PME syndrome characterized by early-onset ataxia, action myoclonus by age 6, scoliosis, and mildly elevated serum creatine kinase. This p.Gly144Trp mutation is equivalent to a loss of function and results in failure of GOSR2 protein to localize to the cis-Golgi.