Dabrafenib Alters MDSC Differentiation and Function by Activation of GCN2.

The effect of targeted therapeutics on anticancer immune responses is poorly understood. The BRAF inhibitor dabrafenib has been reported to activate the integrated stress response (ISR) kinase GCN2, and the therapeutic effect has been partially attributed to GCN2 activation. Because ISR signaling is a key component of myeloid-derived suppressor cell (MDSC) development and function, we measured the effect of dabrafenib on MDSC differentiation and suppressive activity. Our data showed that dabrafenib attenuated MDSC ability to suppress T-cell activity, which was associated with a GCN2-dependent block of the transition from monocytic progenitor to polymorphonuclear (PMN)-MDSCs and proliferative arrest resulting in PMN-MDSC loss. Transcriptional profiling revealed that dabrafenib-driven GCN2 activation altered metabolic features in MDSCs enhancing oxidative respiration, and attenuated transcriptional programs required for PMN development. Moreover, we observed a broad downregulation of transcriptional networks associated with PMN developmental pathways, and increased activity of transcriptional regulons driven by Atf5, Mafg, and Zbtb7a. This transcriptional program alteration underlies the basis for PMN-MDSC developmental arrest, skewing immature MDSC development toward monocytic lineage cells. In vivo, we observed a pronounced reduction in PMN-MDSCs in dabrafenib-treated tumor-bearing mice suggesting that dabrafenib impacts MDSC populations systemically and locally, in the tumor immune infiltrate. Thus, our data reveal transcriptional networks that govern MDSC developmental programs, and the impact of GCN2 stress signaling on the innate immune landscape in tumors, providing novel insight into potentially beneficial off-target effects of dabrafenib. SIGNIFICANCE: An important, but poorly understood, aspect of targeted therapeutics for cancer is the effect on antitumor immune responses. This article shows that off-target effects of dabrafenib activating the kinase GCN2 impact MDSC development and function reducing PMN-MDSCs in vitro and in vivo. This has important implications for our understanding of how this BRAF inhibitor impacts tumor growth and provides novel therapeutic target and combination possibilities.

Dabrafenib alters MDSC differentiation and function by activation of GCN2.

The effect of targeted therapeutics on anti-cancer immune responses is poorly understood. The BRAF inhibitor dabrafenib has been reported to activate the integrated stress response (ISR) kinase GCN2, and the therapeutic effect has been partially attributed to GCN2 activation. Since ISR signaling is a key component of myeloid-derived suppressor cell (MDSC) development and function, we measured the effect of dabrafenib on MDSC differentiation and suppressive activity. Our data showed that dabrafenib attenuated MDSC ability to suppress T cell activity, which was associated with a GCN2-dependent block of the transition from monocytic progenitor to polymorphonuclear (PMN)-MDSCs and proliferative arrest resulting in PMN-MDSC loss. Transcriptional profiling revealed that dabrafenib-driven GCN2 activation altered metabolic features in MDSCs enhancing oxidative respiration, and attenuated transcriptional programs required for PMN development. Moreover, we observed a broad downregulation of transcriptional networks associated with PMN developmental pathways, and increased activity of transcriptional regulons driven by Atf5 , Mafg , and Zbtb7a . This transcriptional program alteration underlies the basis for PMN-MDSC developmental arrest, skewing immature MDSC development towards monocytic lineage cells. In vivo , we observed a pronounced reduction in PMN-MDSCs in dabrafenib-treated tumor-bearing mice suggesting that dabrafenib impacts MDSC populations systemically and locally, in the tumor immune infiltrate. Thus, our data reveals transcriptional networks that govern MDSC developmental programs, and the impact of GCN2 stress signaling on the innate immune landscape in tumors, providing novel insight into potentially beneficial off target effects of dabrafenib.

The transcription factor IRF2 drives interferon-mediated CD8+ T cell exhaustion to restrict anti-tumor immunity.

Type I and II interferons (IFNs) stimulate pro-inflammatory programs that are critical for immune activation, but also induce immune-suppressive feedback circuits that impede control of cancer growth. Here, we sought to determine how these opposing programs are differentially induced. We demonstrated that the transcription factor interferon regulatory factor 2 (IRF2) was expressed by many immune cells in the tumor in response to sustained IFN signaling. CD8+ T cell-specific deletion of IRF2 prevented acquisition of the T cell exhaustion program within the tumor and instead enabled sustained effector functions that promoted long-term tumor control and increased responsiveness to immune checkpoint and adoptive cell therapies. The long-term tumor control by IRF2-deficient CD8+ T cells required continuous integration of both IFN-I and IFN-II signals. Thus, IRF2 is a foundational feedback molecule that redirects IFN signals to suppress T cell responses and represents a potential target to enhance cancer control.

Tryptophan-derived microbial metabolites activate the aryl hydrocarbon receptor in tumor-associated macrophages to suppress anti-tumor immunity.

The aryl hydrocarbon receptor (AhR) is a sensor of products of tryptophan metabolism and a potent modulator of immunity. Here, we examined the impact of AhR in tumor-associated macrophage (TAM) function in pancreatic ductal adenocarcinoma (PDAC). TAMs exhibited high AhR activity and Ahr-deficient macrophages developed an inflammatory phenotype. Deletion of Ahr in myeloid cells or pharmacologic inhibition of AhR reduced PDAC growth, improved efficacy of immune checkpoint blockade, and increased intra-tumoral frequencies of IFNγ+CD8+ T cells. Macrophage tryptophan metabolism was not required for this effect. Rather, macrophage AhR activity was dependent on Lactobacillus metabolization of dietary tryptophan to indoles. Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα+IFNγ+CD8+ T cells; provision of dietary indoles blocked this effect. In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype, suggesting conservation of this regulatory axis in human disease.

GCN2 drives macrophage and MDSC function and immunosuppression in the tumor microenvironment.

General control nonderepressible 2 (GCN2) is an environmental sensor controlling transcription and translation in response to nutrient availability. Although GCN2 is a putative therapeutic target for immuno-oncology, its role in shaping the immune response to tumors is poorly understood. Here, we used mass cytometry, transcriptomics, and transcription factor-binding analysis to determine the functional impact of GCN2 on the myeloid phenotype and immune responses in melanoma. We found that myeloid-lineage deletion of GCN2 drives a shift in the phenotype of tumor-associated macrophages and myeloid-derived suppressor cells (MDSCs) that promotes antitumor immunity. Time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing showed that this was due to changes in the immune microenvironment with increased proinflammatory activation of macrophages and MDSCs and interferon-γ expression in intratumoral CD8+ T cells. Mechanistically, GCN2 altered myeloid function by promoting increased translation of the transcription factor CREB-2/ATF4, which was required for maturation and polarization of macrophages and MDSCs in both mice and humans, whereas targeting Atf4 by small interfering RNA knockdown reduced tumor growth. Last, analysis of patients with cutaneous melanoma showed that GCN2-dependent transcriptional signatures correlated with macrophage polarization, T cell infiltrates, and overall survival. Thus, these data reveal a previously unknown dependence of tumors on myeloid GCN2 signals for protection from immune attack.

Analysis of the HLA-DR peptidome from human dendritic cells reveals high affinity repertoires and nonconventional pathways of peptide generation.

Dendritic cells (DCs) are the major professional APCs of the immune system; however, their MHC-II-associated peptide repertoires have been hard to analyze, mostly because of their scarce presence in blood and tissues. In vitro matured human monocyte-derived DCs (MoDCs) are widely used as professional APCs in experimental systems. In this work, we have applied mass spectrometry to identify the HLA-DR-associated self-peptide repertoires from small numbers of mature MoDCs (∼5 × 106 cells), derived from 7 different donors. Repertoires of 9 different HLA-DR alleles were defined from analysis of 1319 peptides, showing the expected characteristics of MHC-II-associated peptides. Most peptides identified were predicted high binders for their respective allele, formed nested sets, and belonged to endo-lysosomal pathway-degraded proteins. Approximately 20% of the peptides were derived from cytosolic and nuclear proteins, a recurrent finding in HLA-DR peptide repertoires. Of interest, most of these peptides corresponded to single sequences, did not form nested sets, and were located at the C terminus of the parental protein, which suggested alternative processing. Analysis of cleavage patterns for terminal peptides predominantly showed aspartic acid before the cleavage site of both C- and N-terminal peptides and proline immediately after the cleavage site in C-terminal peptides. Proline was also frequent next to the cut sites of internal peptides. These data provide new insights into the Ag processing capabilities of DCs. The relevance of these processing pathways and their contribution to response to infection, tolerance induction, or autoimmunity deserve further analysis.

Central T cell tolerance: Identification of tissue-restricted autoantigens in the thymus HLA-DR peptidome.

Promiscuous gene expression (pGE) of tissue-restricted self-antigens (TRA) in medullary thymic epithelial cells (mTECs) is in part driven by the Autoimmune Regulator gene (AIRE) and essential for self-tolerance. The link between AIRE functional mutations and multi-organ autoimmunity in human and mouse supports the role of pGE. Deep sequencing of the transcriptome revealed that mouse mTECs potentially transcribe an unprecedented range of >90% of all genes. Yet, it remains unclear to which extent these low-level transcripts are actually translated into proteins, processed and presented by thymic APCs to induce tolerance. To address this, we analyzed the HLA-DR-associated thymus peptidome. Within a large panel of peptides from abundant proteins, two TRA peptides were identified: prostate-specific semenogelin-1 (an autoantigen in autoimmune chronic prostatitis/chronic pelvic pain syndrome) and central nervous system-specific contactin-2 (an autoantigen in multiple sclerosis). Thymus expression of both genes was restricted to mTECs. SEMG1 expression was confined to mature HLA-DR(hi) mTECs of male and female donors and was AIRE-dependent, whereas CNTN2 was apparently AIRE-independent and was expressed by both populations of mTECs. Our findings establish a link between pGE, MHC-II peptide presentation and autoimmunity for bona fide human TRAs.

Experience in retake analysis for digital mammography at a university hospital.

Data from one digital mammograph (flat detector active area of 19.2 × 23 cm(2)) were collected over a 1-year period using locally developed software in order to evaluate retakes, their rates, their causes and the possible measures to reduce their occurrence. Among them, 7.1 % of the images were marked as repetitions, and in 16 % of the studies, at least one image was repeated. When evaluating causes of retakes, the primary cause was incorrect positioning (49 %), closely followed by additional retakes in cases of large breasts (44 %). When dealing with large breasts and using a small flat panel, additional images were necessary to fully visualise the breast, and as a consequence, some breast regions received repeated radiation exposure. Moreover, a small detector increases retakes in breasts slightly wrongly positioned. To try and reduce the retake rate, it is important to plan training sessions based on images selected from the retake analysis.

Divergent paths for the selection of immunodominant epitopes from distinct antigenic sources.

Immunodominant epitopes are few selected epitopes from complex antigens that initiate T-cell responses. Here to provide further insights into this process, we use a reductionist cell-free antigen-processing system composed of defined components. We use the system to characterize steps in antigen processing of pathogen-derived proteins or autoantigens and we find distinct paths for peptide processing and selection. Autoantigen-derived immunodominant epitopes are resistant to digestion by cathepsins, whereas pathogen-derived epitopes are sensitive. Sensitivity to cathepsins enforces capture of pathogen-derived epitopes by major histocompatibility complex class II (MHC class II) before processing, and resistance to HLA-DM-mediated-dissociation preserves the longevity of those epitopes. We show that immunodominance is established by higher relative abundance of the selected epitopes, which survive cathepsin digestion either by binding to MHC class II and resisting DM-mediated-dissociation, or being chemically resistant to cathepsins degradation. Non-dominant epitopes are sensitive to both DM and cathepsins and are destroyed.

Composition of the HLA-DR-associated human thymus peptidome.

Major histocompatibility complex class II (MHC-II) molecules bind to and display antigenic peptides on the surface of antigen-presenting cells (APCs). In the absence of infection, MHC-II molecules on APCs present self-peptides and interact with CD4(+) T cells to maintain tolerance and homeostasis. In the thymus, self-peptides bind to MHC-II molecules expressed by defined populations of APCs specialised for the different steps of T-cell selection. Cortical epithelial cells present peptides for positive selection, whereas medullary epithelial cells and dendritic cells are responsible for peptide presentation for negative selection. However, few data are available on the peptides presented by MHC molecules in the thymus. Here, we apply mass spectrometry to analyse and identify MHC-II-associated peptides from five fresh human thymus samples. The data show a diverse self-peptide repertoire, mostly consisting of predicted MHC-II high binders. Despite technical limitations preventing single cell population analyses of peptides, these data constitute the first direct assessment of the HLA-II-bound peptidome and provide insight into how this peptidome is generated and how it drives T-cell repertoire formation.

The Repertoires of Peptides Presented by MHC-II in the Thymus and in Peripheral Tissue: A Clue for Autoimmunity?

T-cell tolerance to self-antigens is established in the thymus through the recognition by developing thymocytes of self-peptide-MHC complexes and induced and maintained in the periphery. Efficient negative selection of auto-reactive T cells in the thymus is dependent on the in situ expression of both ubiquitous and tissue-restricted self-antigens and on the presentation of derived peptides. Weak or inadequate intrathymic expression of self-antigens increases the risk to generate an autoimmune-prone T-cell repertoire. Indeed, even small changes of self-antigen expression in the thymus affect negative selection and increase the predisposition to autoimmunity. Together with other mechanisms, tolerance is maintained in the peripheral lymphoid organs via the recognition by mature T cells of a similar set of self-peptides in homeostatic conditions. However, non-lymphoid peripheral tissue, where organ-specific autoimmunity takes place, often have differential functional processes that may lead to the generation of epitopes that are absent or non-presented in the thymus. These putative differences between peptides presented by MHC molecules in the thymus and in peripheral tissues might be a major key to the initiation and maintenance of autoimmune conditions.

Estenosis congénita de la apertura piriforme nasal / Congenital nasal pyriform aperture stenosis

La obstrucción de la vía aérea nasal es una situación potencialmente letal en los neonatos, puesto que son respiradores nasales obligados. La etiología más habitual es la atresia de coanas, pero hay otras entidades, como la estenosis congénita de la apertura piriforme nasal que, a pesar de su rareza, deben considerarse en el diagnóstico diferencial de un neonato que muestra signos y síntomas de compromiso de la vía aérea superior. A propósito de 2 casos de estenosis de la apertura piriforme nasal en nuestro centro se describen sus principales características y se presenta una revisión de la bibliografía (AU)

Obstruction of the nasal airway is potentially lethal in newborns because they are forced to breathe through their noses. The most common cause of obstruction is atresia of the choanae; however, other entities such as congenital nasal pyriform aperture stenosis, although rare, must be considered in the differential diagnosis of a newborn that shows signs and symptoms of upper airway compromise. We report two cases of nasal pyriform aperture stenosis seen at our center; we describe the main characteristics of the condition and review the relevant literature (AU)

Recurrencia en partes blandas de tumor de células gigantes del cúbito / Recurrence of giant-cell cubital tumor in soft tissue

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[Congenital nasal pyriform aperture stenosis]. / Estenosis congénita de la apertura piriforme nasal.

Obstruction of the nasal airway is potentially lethal in newborns because they are forced to breathe through their noses. The most common cause of obstruction is atresia of the choanae; however, other entities such as congenital nasal pyriform aperture stenosis, although rare, must be considered in the differential diagnosis of a newborn that shows signs and symptoms of upper airway compromise. We report two cases of nasal pyriform aperture stenosis seen at our center; we describe the main characteristics of the condition and review the relevant literature.

[Cystic lymphangioma of the spleen in a patient with Klippel-Trenaunay-Weber syndrome: MRI findings]. / Linfangioma quístico esplénico en una paciente con síndrome de Klippel-Trenaunay-Weber. Hallazgos en la ecografía y la RM.

We present the case of a 22-year-old woman with Klippel-Trenaunay-Weber syndrome who presented with clinical signs and symptoms of gastrointestinal obstruction. Ultrasonography and magnetic resonance imaging showed massive splenomegaly with multiple cysts replacing the normal parenchyma of the spleen. Histologic study after splenectomy confirmed the diagnosis of cystic lymphangioma of the spleen, which is an exceptional manifestation of Klippel-Trenaunay-Weber syndrome.

Linfangioma quístico esplénico en una paciente con síndrome de Klippel-Trenaunay-Weber. Hallazgos en la ecografía y la RM / Cystic lymphangioma of the soleen in a patient with Klippel-Trenaunay- Weber syndrome: MRI findings

Presentamos un caso de síndrome de Klippel-Trenaunay-Weber en una mujer de 22 años con clínica obstructiva gastrointestinal. La ecografía y la resonancia magnética mostraron una esplenomegalia masiva con múltiples quistes reemplazando el parénquima esplénico normal.Se realizó esplenectomía y el estudio histológico confirmó el diagnóstico de linfangioma quístico esplénico. Esta entidad es una manifestaciónexcepcional del síndrome de Klippel-Trenaunay-Weber

We present the case of a 22-year-old woman with Klippel-Trenaunay-Weber syndrome who presented with clinical signs and symptoms of gastrointestinal obstruction. Ultrasonography and magnetic resonance imaging showed massive splenomegaly with multiple cysts replacingthe normal parenchyma of the spleen. Histologic study after splenectomy confirmed the diagnosis of cystic lymphangioma of the spleen, which is an exceptional manifestation of Klippel-Trenaunay-Weber syndrome

Neumotórax catamenial. Hallazgos radiológicos / Imaging findings in catamenial pneumothorax

Objetivo. Mostrar la forma de presentación y los hallazgos radiológicos del neumotórax catamenial.Material y método. Se revisaron las pruebas de imagen (radiografía simple, tomografía computarizada [TC] y resonancia magnética [RM]) de 6 mujeres de entre 28 y 44 años que presentaron neumotórax recurrentes asociados a la menstruación. A todas se les realizó cirugía por videotoracoscopia (VTS) y en tres de ellas fue necesaria la realización de una toracotomía debido a la recurrencia del neumotórax.Resultados. En tres casos se realizó TC, encontrando nódulos pleurales en dos casos, uno de ellos confirmado en la RM. Histológicamente se demostró endometriosis pleural en un único caso. Se identificaron agujeros diafragmáticos y bullas en 5 de las 6 pacientes.Discusión. Los síntomas más frecuentes del neumotórax catamenial son el dolor torácico, la disnea y la hemoptisis. La localización más frecuente es el lado derecho (90%). Los hallazgos radiológicos son neumotórax, hemotórax o hidroneumotórax. La TC y la RM pueden ayudar en la identificación de lesiones pleurales no visibles en la radiografía simple que suponen un hallazgo muy frecuente en la cirugía.Conclusión. El diagnóstico de neumotórax catamenial debe sospecharse en mujeres en edad fértil con historia de neumotórax recidivantes coincidentes con la menstruación. La TC y la RM pueden ayudar a identificar lesiones sospechosas de endometriosis

Objective. To show the presentation and imaging findings of catamenial pneumothorax.Material and methods. We reviewed the imaging tests (plain-film radiography, computed tomography [CT], magnetic resonance [MR]) performed in six women aged between 28 and 44 years with recurrent pneumothorax associated to menstruation. All patients underwent videothoracoscopic surgery and thoracotomy was necessary in three due to the recurrence of the pneumothorax.Results. CT was performed in three cases and found pleural nodules in two; one of these was confirmed at MR. Pleural endometriosis was only demonstrated at histological examination in one case. Diaphragmaticblebs and bullae were found in five of the six patients.Discussion. The most common symptoms of catamenial pneumothorax are chest pain, dyspnea, and hemoptysis. The right side is affected in 90% of cases. The radiological findings are pneumothorax, hemothorax,or hydropneumothorax. CT and MR can help to identify thepleural lesions that are not visible on plain-film radiographs and are a very common finding at surgery.Conclusion. The diagnosis of catamenial pneumothorax should be suspected in fertile-aged women with a history of recurrent pneumothorax coinciding with menstruation. CT and MR can help to identify lesions suspicious of endometriosis

[Imaging findings in catamenial pneumothorax]. / Neumotórax catamenial.Hallazgos radiológicos.

OBJECTIVE: To show the presentation and imaging findings of catamenial pneumothorax. MATERIAL AND METHODS: We reviewed the imaging tests (plain-film radiography, computed tomography [CT], magnetic resonance [MR]) performed in six women aged between 28 and 44 years with recurrent pneumothorax associated to menstruation. All patients underwent videothoracoscopic surgery and thoracotomy was necessary in three due to the recurrence of the pneumothorax. RESULTS: CT was performed in three cases and found pleural nodules in two; one of these was confirmed at MR. Pleural endometriosis was only demonstrated at histological examination in one case. Diaphragmatic blebs and bullae were found in five of the six patients. DISCUSSION: The most common symptoms of catamenial pneumothorax are chest pain, dyspnea, and hemoptysis. The right side is affected in 90% of cases. The radiological findings are pneumothorax, hemothorax, or hydropneumothorax. CT and MR can help to identify the pleural lesions that are not visible on plain-film radiographs and are a very common finding at surgery. CONCLUSION: The diagnosis of catamenial pneumothorax should be suspected in fertile-aged women with a history of recurrent pneumothorax coinciding with menstruation. CT and MR can help to identify lesions suspicious of endometriosis.