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Project-based learning (PBL) is a student-centered approach that allows students to build on prior knowledge and address relevant problems while working on challenging projects. PBL is well-suited to undergraduate neuroscience courses because students are often interested in learning about diseased states of the nervous system, but can be discouraged by having to learn the chemical and cellular mechanisms underlying pathologies in a lecture-based learning environment. PBL provides students with a significant learning experience that excites them and can help them learn challenging content. Drawing from the recommendations of multiple STEM education reform efforts, I examined the effectiveness of using PBL in an undergraduate neurobiology course to provide students with significant and engaging learning experiences. Students were grouped into teams using a guild system and completed three substantial projects consisting of team-authored research papers and poster presentations. Each project was designed to address fundamental neuroscience concepts using a real-world problem. By the end of the course, students were more confident in their understanding of neuroscience and had greater understanding of neuroscience concepts. Student attitudes toward working on projects or working as a member of team did not change but remained positive throughout course. Taken together, these results suggest that PBL can be an effective way to actively engage students while allowing them to learn, integrate and communicate core neuroscience concepts.
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Invariant natural killer T cells are a rare, heterogeneous T-cell subset with cytotoxic and immunomodulatory properties. During thymic development, murine invariant natural killer T cells go through different maturation stages differentiating into distinct sublineages, namely, invariant natural killer T1, 2, and 17 cells. Recent reports indicate that invariant natural killer T2 cells display immature properties and give rise to other subsets, whereas invariant natural killer T1 cells seem to be terminally differentiated. Whether human invariant natural killer T cells follow a similar differentiation model is still unknown. To define the maturation stages and assess the sublineage commitment of human invariant natural killer T cells during thymic development, in this study, we performed single-cell RNA sequencing analysis on human Vα24+Vß11+ invariant natural killer T cells isolated from thymocytes. We show that these invariant natural killer T cells displayed heterogeneity, and our unsupervised analysis identified 5 clusters representing different maturation stages, from an immature profile with high expression of genes important for invariant natural killer T cell development and proliferation to a mature, fully differentiated profile with high levels of cytotoxic effector molecules. Evaluation of expression of sublineage-defining gene sets revealed mainly cells with an invariant natural killer T2 signature in the most immature cluster, whereas the more differentiated ones displayed an invariant natural killer T1 signature. Combined analysis with a publicly available single-cell RNA sequencing data set of human invariant natural killer T cells from peripheral blood suggested that the 2 main subsets exist both in thymus and in the periphery, while a third more immature one was restricted to the thymus. Our data point to the existence of different maturation stages of human thymic invariant natural killer T cells and provide evidence for sublineage commitment of invariant natural killer T cells in the human thymus.
Assuntos
Células T Matadoras Naturais , Humanos , Camundongos , Animais , Células T Matadoras Naturais/metabolismo , Timo , Timócitos , Subpopulações de Linfócitos T , Diferenciação Celular/genética , Perfilação da Expressão GênicaRESUMO
Increasing evidence supports the interplay between oncogenic mutations and immune escape mechanisms. Strategies to counteract the immune escape mediated by oncogenic signaling could provide improved therapeutic options for patients with various malignancies. As mutant calreticulin (CALR) is a common driver of myeloproliferative neoplasms (MPN), we analyzed the impact of oncogenic CALRdel52 on the bone marrow (BM) microenvironment in MPN. Single-cell RNA-sequencing revealed that CALRdel52 led to the expansion of TGF-ß1-producing erythroid progenitor cells and promoted the expansion of FoxP3+ regulatory T cells (Treg) in a murine MPN model. Treatment with an anti-TGF-ß antibody improved mouse survival and increased the glycolytic activity in CD4+ and CD8+ T cells in vivo, while T cell depletion abrogated the protective effects conferred by neutralizing TGF-ß. TGF-ß1 reduced perforin and TNF-α production by T cells in vitro. TGF-ß1 production by CALRdel52 cells was dependent on JAK1/2, PI3K, and ERK activity, which activated the transcription factor Sp1 to induce TGF-ß1 expression. In four independent patient cohorts, TGF-ß1 expression was increased in the BM of MPN patients compared to healthy individuals, and the BM of MPN patients contained a higher frequency of Treg compared to healthy individuals. Together, this study identified an ERK/Sp1/TGF-ß1 axis in CALRdel52 MPNs as a mechanism of immunosuppression that can be targeted to elicit T-cell-mediated cytotoxicity.
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Leukemia relapse is a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT). We tested the potential of targeting TIM-3 for improving graft-versus-leukemia (GVL) effects. We observed differential expression of TIM-3 ligands when hematopoietic stem cells overexpressed certain oncogenic-driver mutations. Anti-TIM-3 Ab-treatment improved survival of mice bearing leukemia with oncogene-induced TIM-3 ligand expression. Conversely, leukemia cells with low ligand expression were anti-TIM-3 treatment-resistant. In vitro, TIM-3 blockade or genetic deletion in CD8+ T cells (Tc) enhanced Tc activation, proliferation and IFN-γ production while enhancing GVL effects, preventing Tc exhaustion and improving Tc cytotoxicity and glycolysis in vivo. Conversely, TIM-3 deletion in myeloid cells did not affect allogeneic Tc proliferation and activation in vitro, suggesting that anti-TIM-3-treatment-mediated GVL effects are Tc-induced. In contrast to anti-PD-1 and anti-CTLA-4-treatment, anti-TIM-3-treatment did not enhance acute graft-versus-host-disease (aGVHD). TIM-3 and its ligands were frequently expressed in acute myeloid leukemia (AML) cells of patients with post-allo-HCT relapse. We deciphered the connection between oncogenic mutations found in AML and TIM-3 ligands expression and identify anti-TIM-3-treatment as a strategy to enhance GVL effects via metabolic and transcriptional Tc-reprogramming, without exacerbation of aGVHD. Our findings support clinical testing of anti-TIM-3 Abs in patients with AML relapse post-allo-HCT.
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A substantial proportion of cancer patients do not benefit from platinum-based chemotherapy (CT) due to the emergence of drug resistance. Here, we apply elemental imaging to the mapping of CT biodistribution after therapy in residual colorectal cancer and achieve a comprehensive analysis of the genetic program induced by oxaliplatin-based CT in the tumor microenvironment. We show that oxaliplatin is largely retained by cancer-associated fibroblasts (CAFs) long time after the treatment ceased. We determine that CT accumulation in CAFs intensifies TGF-beta activity, leading to the production of multiple factors enhancing cancer aggressiveness. We establish periostin as a stromal marker of chemotherapeutic activity intrinsically upregulated in consensus molecular subtype 4 (CMS4) tumors and highly expressed before and/or after treatment in patients unresponsive to therapy. Collectively, our study underscores the ability of CT-retaining CAFs to support cancer progression and resistance to treatment.
Assuntos
Antineoplásicos , Fibroblastos Associados a Câncer , Neoplasias Colorretais , Humanos , Fibroblastos Associados a Câncer/patologia , Oxaliplatina/farmacologia , Distribuição Tecidual , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Microambiente Tumoral , Fibroblastos/patologia , Linhagem Celular TumoralRESUMO
Therapy-resistant leukemia stem and progenitor cells (LSC) are a main cause of acute myeloid leukemia (AML) relapse. LSC-targeting therapies may thus improve outcome of patients with AML. Here we demonstrate that LSCs present HLA-restricted antigens that induce T-cell responses allowing for immune surveillance of AML. Using a mass spectrometry-based immunopeptidomics approach, we characterized the antigenic landscape of patient LSCs and identified AML- and AML/LSC-associated HLA-presented antigens absent from normal tissues comprising nonmutated peptides, cryptic neoepitopes, and neoepitopes of common AML driver mutations of NPM1 and IDH2. Functional relevance of shared AML/LSC antigens is illustrated by presence of their cognizant memory T cells in patients. Antigen-specific T-cell recognition and HLA class II immunopeptidome diversity correlated with clinical outcome. Together, these antigens shared among AML and LSCs represent prime targets for T cell-based therapies with potential of eliminating residual LSCs in patients with AML. SIGNIFICANCE: The elimination of therapy-resistant leukemia stem and progenitor cells (LSC) remains a major challenge in the treatment of AML. This study identifies and functionally validates LSC-associated HLA class I and HLA class II-presented antigens, paving the way to the development of LSC-directed T cell-based immunotherapeutic approaches for patients with AML. See related commentary by Ritz, p. 430 . This article is featured in Selected Articles from This Issue, p. 419.
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Antígenos HLA , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/genética , Peptídeos , Células-TroncoRESUMO
About 50% of human epidermal growth factor receptor 2 (HER2)+ breast cancer patients do not benefit from HER2-targeted therapy and almost 20% of them relapse after treatment. Here, we conduct a detailed analysis of two independent cohorts of HER2+ breast cancer patients treated with trastuzumab to elucidate the mechanisms of resistance to anti-HER2 monoclonal antibodies. In addition, we develop a fully humanized immunocompetent model of HER2+ breast cancer recapitulating ex vivo the biological processes that associate with patients' response to treatment. Thanks to these two approaches, we uncover a population of TGF-beta-activated cancer-associated fibroblasts (CAF) specific from tumors resistant to therapy. The presence of this cellular subset related to previously described myofibroblastic (CAF-S1) and podoplanin+ CAF subtypes in breast cancer associates with low IL2 activity. Correspondingly, we find that stroma-targeted stimulation of IL2 pathway in unresponsive tumors restores trastuzumab anti-cancer efficiency. Overall, our study underscores the therapeutic potential of exploiting the tumor microenvironment to identify and overcome mechanisms of resistance to anti-cancer treatment.
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Neoplasias da Mama , Fibroblastos Associados a Câncer , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Feminino , Humanos , Fatores Imunológicos , Imunoterapia , Interleucina-2 , Receptor ErbB-2 , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Microambiente TumoralRESUMO
The DNAJB1-PRKACA fusion transcript is the oncogenic driver in fibrolamellar hepatocellular carcinoma, a lethal disease lacking specific therapies. This study reports on the identification, characterization, and immunotherapeutic application of HLA-presented neoantigens specific for the DNAJB1-PRKACA fusion transcript in fibrolamellar hepatocellular carcinoma. DNAJB1-PRKACA-derived HLA class I and HLA class II ligands induce multifunctional cytotoxic CD8+ and T-helper 1 CD4+ T cells, and their cellular processing and presentation in DNAJB1-PRKACA expressing tumor cells is demonstrated by mass spectrometry-based immunopeptidome analysis. Single-cell RNA sequencing further identifies multiple T cell receptors from DNAJB1-PRKACA-specific T cells. Vaccination of a fibrolamellar hepatocellular carcinoma patient, suffering from recurrent short interval disease relapses, with DNAJB1-PRKACA-derived peptides under continued Poly (ADP-ribose) polymerase inhibitor therapy induces multifunctional CD4+ T cells, with an activated T-helper 1 phenotype and high T cell receptor clonality. Vaccine-induced DNAJB1-PRKACA-specific T cell responses persist over time and, in contrast to various previous treatments, are accompanied by durable relapse free survival of the patient for more than 21 months post vaccination. Our preclinical and clinical findings identify the DNAJB1-PRKACA protein as source for immunogenic neoepitopes and corresponding T cell receptors and provide efficacy in a single-patient study of T cell-based immunotherapy specifically targeting this oncogenic fusion.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patologia , Regulação Neoplásica da Expressão Gênica , Recidiva Local de Neoplasia/genética , Proteínas de Fusão Oncogênica/genética , Imunoterapia , Peptídeos/genética , Proteínas de Choque Térmico HSP40/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP CíclicoRESUMO
Inherited bone marrow failure syndromes (IBMFSs) are a heterogeneous group of disorders characterized by defective hematopoiesis, impaired stem cell function, and cancer susceptibility. Diagnosis of IBMFS presents a major challenge due to the large variety of associated phenotypes, and novel, clinically relevant biomarkers are urgently needed. Our study identified nuclear interaction partner of ALK (NIPA) as an IBMFS gene, as it is significantly downregulated in a distinct subset of myelodysplastic syndrome-type (MDS-type) refractory cytopenia in children. Mechanistically, we showed that NIPA is major player in the Fanconi anemia (FA) pathway, which binds FANCD2 and regulates its nuclear abundance, making it essential for a functional DNA repair/FA/BRCA pathway. In a knockout mouse model, Nipa deficiency led to major cell-intrinsic defects, including a premature aging phenotype, with accumulation of DNA damage in hematopoietic stem cells (HSCs). Induction of replication stress triggered a reduction in and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the knockout mice with 100% penetrance. Taken together, the results of our study add NIPA to the short list of FA-associated proteins, thereby highlighting its potential as a diagnostic marker and/or possible target in diseases characterized by hematopoietic failure.
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Síndrome Congênita de Insuficiência da Medula Óssea , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Células-Tronco Hematopoéticas/metabolismo , Proteínas Nucleares , Animais , Síndrome Congênita de Insuficiência da Medula Óssea/genética , Síndrome Congênita de Insuficiência da Medula Óssea/metabolismo , Síndrome Congênita de Insuficiência da Medula Óssea/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Células-Tronco Hematopoéticas/patologia , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Ligação ProteicaRESUMO
Unintentional plagiarism frequently occurs in undergraduate writing assignments because students are unaware of the complexity of correct paraphrasing and citation rules. There is often a lack of formal instruction in science courses on proper paraphrasing and citation to reduce plagiarism. To address this deficit, we developed a brief activity to teach students to recognize the range of paraphrasing and citation errors that can result in plagiarism. The activity was used in a biology-focused scientific literacy course, but it can be incorporated into different instructional settings, with undergraduate students of all levels. During this classroom activity, part 1 addresses the nuances associated with proper paraphrasing and citation in scientific writing and part 2 asks students to practice paraphrasing and properly citing a passage from a scientific source. Pretest results revealed that students were proficient at identifying plagiarism when a citation error occurred but were less proficient at recognizing improper paraphrasing (patchwriting or direct plagiarism). Posttest results indicated that the activity was effective at increasing the students' ability to recognize a paraphrasing error even when a correct citation was present. Students also reported higher confidence in their understanding of what constitutes plagiarism and that they are more confident in their ability to properly paraphrase and cite scientific source content.
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Sensory neurons in aging mammals undergo changes in anatomy, physiology and gene expression that correlate with reduced sensory perception. In this study we compared young and aged mice to identify proteins that might contribute to this loss of sensation. We first show using behavioral testing that thermal sensitivity in aged male and female mice is reduced. Expression of sodium channel (Nav1.8 and Nav1.9) and transient receptor potential vanilloid (TRPV) channels in DRG and peripheral nerves of young and old male mice was then examined. Immunoblotting and RT-PCR assays showed reduced Nav1.8 levels in aged mice. No change was measured in TRPV1 mRNA levels in DRG though TRPV1 protein appeared reduced in the DRG and peripheral nerves. The GFRalpha3 receptor, which binds the growth factor artemin and is expressed by TRPV1-positive neurons, was also decreased in the DRG of aged animals. These findings indicate that loss of thermal sensitivity in aging animals may result from a decreased level of TRPV1 and Nav1.8 and decreased trophic support that inhibits efficient transport of channel proteins to peripheral afferents.
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Envelhecimento/fisiologia , Expressão Gênica/fisiologia , Neurônios Aferentes/fisiologia , Canais de Sódio/metabolismo , Canais de Cátion TRPV/metabolismo , Sensação Térmica/fisiologia , Fatores Etários , Animais , Comportamento Animal , Western Blotting/métodos , Feminino , Gânglios Espinais/citologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Imuno-Histoquímica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.8 , Fibras Nervosas/metabolismo , RNA Mensageiro/biossíntese , Tempo de Reação/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores Sexuais , Canais de Sódio/genética , Canais de Cátion TRPV/genéticaRESUMO
Most, if not all, nociceptor sensory neurons are dependent on nerve growth factor (NGF) during early embryonic development. A large subpopulation of these sensory neurons loses NGF dependency between embryonic day 16 and postnatal day 14 and become responsive to glial cell line-derived growth factor (GDNF), a member of the transforming growth factor beta (TGF-beta) family. To examine the survival and phenotypic effects of GDNF on sensory neurons in vivo, we generated transgenic mice that overexpress GDNF in the skin. GDNF-overexpresser mice had increased numbers of small unmyelinated sensory neurons that express the tyrosine kinase receptor Ret and bind the plant isolectin B4 (IB4). Surprisingly, in wild-type and transgenic mice, few ( approximately 2%) IB4-positive neurons expressed the vanilloid receptor VR1, a heat-sensitive receptor expressed by many IB4-positive neurons of the rat. Thus, in mouse, GDNF-dependent IB4-positive neurons must use a non-VR1 heat receptor. In addition, the behavior of GDNF-overexpresser animals to noxious heat or mechanical stimuli was indistinguishable from wild-type animals, indicating that, on a behavioral level, peripherally applied GDNF does not alter the sensitivity of the somatosensory system.
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Lectinas/metabolismo , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/biossíntese , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/metabolismo , Receptores de Droga/biossíntese , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Comportamento Animal , Peptídeo Relacionado com Gene de Calcitonina/biossíntese , Diferenciação Celular , Sobrevivência Celular/efeitos dos fármacos , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Temperatura Alta , Humanos , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/farmacologia , Neurônios Aferentes/citologia , Medição da Dor , Limiar da Dor/fisiologia , Nervos Periféricos/citologia , Nervos Periféricos/fisiologia , Estimulação Física , RNA Mensageiro/biossíntese , Receptores Purinérgicos P2/biossíntese , Receptores Purinérgicos P2X3 , Pele/inervação , Canais de Cátion TRPV , TransgenesRESUMO
Presynaptic synthesis of acetylcholine (ACh) requires a steady supply of choline, acquired by a plasma membrane, hemicholinium-3-sensitive (HC-3) choline transporter (CHT). A significant fraction of synaptic choline is recovered from ACh hydrolyzed by acetylcholinesterase (AChE) after vesicular release. Although antecedent neuronal activity is known to dictate presynaptic CHT activity, the mechanisms supporting this regulation are unknown. We observe an exclusive localization of CHT to cholinergic neurons and demonstrate that the majority of CHTs reside on small vesicles within cholinergic presynaptic terminals in the rat and mouse brain. Furthermore, immunoisolation of presynaptic vesicles with multiple antibodies reveals that CHT-positive vesicles carry the vesicular acetylcholine transporter (VAChT) and synaptic vesicle markers such as synaptophysin and Rab3A and also contain acetylcholine. Depolarization of synaptosomes evokes a Ca2+-dependent botulinum neurotoxin C-sensitive increase in the Vmax for HC-3-sensitive choline uptake that is accompanied by an increase in the density of CHTs in the synaptic plasma membrane. Our study leads to the novel hypothesis that CHTs reside on a subpopulation of synaptic vesicles in cholinergic terminals that can transit to the plasma membrane in response to neuronal activity to couple levels of choline re-uptake to the rate of ACh release.
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Proteínas de Membrana Transportadoras/metabolismo , Terminações Pré-Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Animais , Especificidade de Anticorpos , Biomarcadores/análise , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Fibras Colinérgicas/química , Fibras Colinérgicas/metabolismo , Técnicas de Imunoadsorção , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/química , Neurônios/metabolismo , Células PC12 , Terminações Pré-Sinápticas/química , Transporte Proteico , Ratos , Frações Subcelulares/química , Vesículas Sinápticas/química , Vesículas Sinápticas/classificação , Proteínas Vesiculares de Transporte de AcetilcolinaRESUMO
Vanilloid receptor 1 (TRPV1) has been proposed to be the principal heat-responsive channel for nociceptive neurons. The skin of both rat and mouse receives major projections from primary sensory afferents that bind the plant lectin isolectin B4 (IB4). The majority of IB4-positive neurons are known to be heat-responsive nociceptors. Previous studies suggested that, unlike rat, mouse IB4-positive cutaneous afferents did not express TRPV1 immunoreactivity. Here, multiple antisera were used to confirm that mouse and rat have different distributions of TRPV1 and that TRPV1 immunoreactivity is absent in heat-sensitive nociceptors. Intracellular recording in TRPV1(-/-) mice was then used to confirm that TRPV1 was not required for detecting noxious heat. TRPV1(-/-) mice had more heat-sensitive neurons, and these neurons had normal temperature thresholds and response properties. Moreover, in TRPV1(-/-) mice, 82% of heat-responsive neurons did not express immunoreactivity for TRPV2, another putative noxious heat channel.
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Canais de Cálcio/deficiência , Gânglios Espinais/citologia , Temperatura Alta , Canais Iônicos/deficiência , Neurônios Aferentes/química , Nociceptores/fisiologia , Receptores de Droga/deficiência , Sequência de Aminoácidos , Animais , Canais de Cálcio/genética , Canais de Cálcio/fisiologia , Epiderme/fisiologia , Feminino , Canais Iônicos/genética , Canais Iônicos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Neurônios Aferentes/fisiologia , Nociceptores/química , Lectinas de Plantas/análise , Ratos , Ratos Sprague-Dawley , Receptores de Droga/genética , Receptores de Droga/fisiologia , Especificidade da Espécie , Canais de Cátion TRPVRESUMO
At least two classes of neciceptors can be distinguished based on their growth factor requirements: glial cell-line derived neurotrophic factor (GDNF)- and nerve growth factor (NGF)-dependent primary afferent neurons. Based on numerous anatomical and biochemical differences, GDNF- and NGF-dependent neurons have been proposed to be involved in the development of different types of persistent pain. To examine this hypothesis we used two lines of transgenic mice that contained a supernormal number of either NGF- or GDNF-dependent neurons (referred to as NGF-OE and GDNF-OE mice, respectively). These mice were tested in a model of inflammatory pain (induced by injection of complete Freund's adjuvant) and neuropathic pain (using a spinal nerve ligation protocol). Contrary to expectations, neither line of transgenic mice became more hyperalgesic following induction of persistent pain. In fact, NGF-OE mice recovered more rapidly and became hypoalgesic despite extensive paw swelling in the inflammatory pain model. In the neuropathic pain model, only wildtype mice became hyperalgesic. Real-time PCR analysis showed that the NGF-OE and GDNF-OE mice exhibited changes in neuronal-specific mRNAs in the dorsal root ganglia but not the spinal cord dorsal horn. These results indicate that increasing the number of nociceptors results in potent compensatory mechanisms that may begin with changes in the sensory neurons themselves.