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Regulatory T (Treg) cells maintain immune homeostasis and prevent inflammatory and autoimmune responses. During development, thymocytes bearing a moderately self-reactive T cell receptor (TCR) can be selected to become Treg cells. Several observations suggest that also in the periphery mature Treg cells continuously receive self-reactive TCR signals. However, the importance of this inherent autoreactivity for Treg cell biology remains poorly defined. To address this open question, we genetically ablated the TCR of mature Treg cells in vivo. These experiments revealed that TCR-induced Treg lineage-defining Foxp3 expression and gene hypomethylation were uncoupled from TCR input in mature Treg cells. However, Treg cell homeostasis, cell-type-specific gene expression and suppressive function critically depend on continuous triggering of their TCR.
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Autoimunidade/imunologia , Fatores de Transcrição Forkhead/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T Reguladores/imunologia , Animais , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Metilação de DNA/imunologia , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Forkhead/genética , Inflamação/imunologia , Fatores Reguladores de Interferon/biossíntese , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/metabolismo , Timócitos/citologiaRESUMO
Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy. However, structured knowledge to mitigate a patient's specific risk of developing adverse events are limited. Nevertheless, there is an exponential growth of clinical studies combining conventional therapies such as radiation therapy (RT) with ICIs. Cutaneous reactions are among the most common adverse events after monotherapy with either ICIs or RT. So far, little is known about interindividual differences for the risk of developing severe tissue toxicity after the combination of RT with ICIs, and the underlying biological mechanisms are ill defined. We used experimental models of RT-induced skin injury to analyze skin toxicity after simultaneous application of ICIs. We compared different RT regimens such as fractionated or stereotactic RT with varying dose intensity. Strikingly, we found that simultaneous application of RT and ICIs did not significantly aggravate acute skin injury in two different mouse strains. Detailed examination of long-term tissue damage of the skin revealed similar signs of epidermal hyperplasia, dermal fibrosis, and adnexal atrophy. In summary, we here present the first experimental study demonstrating the excellent safety profiles of concurrent treatment with RT and ICIs. These findings will help to interpret the development of adverse events of the skin after radioimmunotherapy and guide the design of new clinical trials and clinical decision-making in individual cases. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Inibidores de Checkpoint Imunológico , Dermatopatias , Animais , Camundongos , Pele , Reino UnidoRESUMO
Patients with X-linked lymphoproliferative syndrome type 2 (XLP-2) (BIRC4 deficiency) suffer from hyperinflammation often observed during the conditioning regimen prior to allogeneic bone marrow transplant. This article shows that in mice hematopoietic recipient cells contribute to graft-versus-host disease by the secretion of elevated levels of proinflammatory cytokines during engraftment when BIRC4 is absent.
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Doenças Genéticas Ligadas ao Cromossomo X/terapia , Doença Enxerto-Hospedeiro/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Transtornos Linfoproliferativos/terapia , Linfócitos T/metabolismo , Doadores de Tecidos , Animais , Transplante de Medula Óssea/métodos , Citocinas/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Sobrevivência de Enxerto , Doença Enxerto-Hospedeiro/genética , Humanos , Mediadores da Inflamação/metabolismo , Proteínas Inibidoras de Apoptose/deficiência , Proteínas Inibidoras de Apoptose/genética , Ativação Linfocitária , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transplante HomólogoRESUMO
NKT cells represent a small subset of glycolipid-recognizing T cells that are heavily implicated in human allergic, autoimmune, and malignant diseases. In the thymus, precursor cells recognize self-glycolipids by virtue of their semi-invariant TCR, which triggers NKT cell lineage commitment and maturation. During their development, NKT cells are polarized into the NKT1, NKT2, and NKT17 subsets, defined through their cytokine-secretion patterns and the expression of key transcription factors. However, we have largely ignored how the differentiation into the NKT cell subsets is regulated. In this article, we describe the mRNA-binding Roquin-1 and -2 proteins as central regulators of murine NKT cell fate decisions. In the thymus, T cell-specific ablation of the Roquin paralogs leads to a dramatic expansion of NKT17 cells, whereas peripheral mature NKT cells are essentially absent. Roquin-1/2-deficient NKT17 cells show exaggerated lineage-specific expression of nearly all NKT17-defining proteins tested. We show through mixed bone marrow chimera experiments that NKT17 polarization is mediated through cell-intrinsic mechanisms early during NKT cell development. In contrast, the loss of peripheral NKT cells is due to cell-extrinsic factors. Surprisingly, Roquin paralog-deficient NKT cells are, in striking contrast to conventional T cells, compromised in their ability to secrete cytokines. Altogether, we show that Roquin paralogs regulate the development and function of NKT cell subsets in the thymus and periphery.
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Diferenciação Celular/imunologia , Células T Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Citometria de Fluxo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The NF-κB regulator A20 limits inflammation by providing negative feedback in myeloid cells and B cells. Functional lack of A20 has been linked to several inflammatory and autoimmune diseases. To define how A20 affects the functionality of T effector cells in a highly inflammatory environment, we performed conventional allogeneic hematopoietic stem cell transplantation (allo-HSCT) with A20-deficient CD4+ and CD8+ donor T cells in mice. Severity and mortality of graft-versus-host disease (GVHD) after allo-HSCT was drastically reduced in recipients transplanted with conventional doses of A20-deficient T cells. Consistently, we found that the A20-deficient donor T-cell compartment was strongly diminished at various timepoints after allo-HSCT. However, proportionally more A20-deficient donor T cells produced IFN-γ and systemic inflammation was elevated early after allo-HSCT. Consequently, increasing the dose of transplanted A20-deficient T cells reversed the original phenotype and resulted in enhanced GVHD mortality compared to recipients that received A20+/+ T cells. Still, A20-deficient T cells, activated either through T cell receptor-dependent or -independent mechanisms, were less viable than control A20+/+ T cells, highlighting that A20 balances both, T-cell activation and survival. Thus, our findings suggest that targeting A20 in T cells may allow to modulate T-cell-mediated inflammatory diseases like GVHD.
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Doença Enxerto-Hospedeiro/imunologia , Linfócitos T/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/imunologia , Animais , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante HomólogoRESUMO
Activation of the C-type lectin receptor Dectin-1 by ß-glucans triggers multiple signals within DCs that result in activation of innate immunity. While these mechanisms can potently prime CD8+ cytotoxic T-cell (CTL) responses without additional adjuvants, the Dectin-1 effector pathways that control CTL induction remain unclear. Here we demonstrate that Dectin-1-induced CTL cross-priming in mice does not require inflammasome activation but strictly depends on the adapter protein Card9 in vitro. In vivo, Dectin-1-mediated Card9 activation after vaccination drives both expansion and activation of Ag-specific CTLs, resulting in long-lasting CTL responses that are sufficient to protect mice from tumor challenge. This Dectin-1-induced antitumor immune response was independent of NK cell function and completely abrogated in Card9-deficient mice. Thus, our results demonstrate that Dectin-1-triggered Card9 signaling but not inflammasome activation can potently cross-prime Ag-specific CTLs, suggesting that this pathway would be a candidate for immunotherapy and vaccine development.
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Proteínas Adaptadoras de Sinalização CARD/metabolismo , Lectinas Tipo C/metabolismo , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Proteínas Adaptadoras de Sinalização CARD/deficiência , Proteínas Adaptadoras de Sinalização CARD/genética , Apresentação Cruzada , Imunidade Inata , Inflamassomos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/fisiopatologia , Transdução de Sinais , VacinaçãoRESUMO
Background: Most clinical studies failed to elicit a strong antitumor immune response and subsequent systemic tumor regression after radiation therapy (RT), even in combination with the immune checkpoint inhibitors (ICI) anti-CTLA4 or anti-PD1. Mechanistically, type I interferon (IFN-I) activation is essential for the development of such abscopal effects (AE); however, mechanisms driving or limiting IFN-I activation are ill defined. Groundbreaking discoveries have shown that antibiotics (ABx) can affect oncological outcomes and that microbiota-derived metabolites can modulate systemic antitumor immunity. Recent studies have demonstrated that the bacterial metabolites desaminotyrosine (DAT) and indole-3-carboxaldehyde (ICA) can enhance IFN-I activation in models of inflammatory diseases. Materials and Methods: The subcutaneous bilateral MC38 tumor model is a widely used experimental tool to study the AE in mice. We applied it to explore the influence of broad-spectrum ABx, DAT and ICA on the AE after radioimmunotherapy (RIT). We performed 1x8 Gy of the primary tumor ± anti-CTLA4 or anti-PD1, and ± daily oral application of ABx or metabolites. Result: Combinatory ABx had neither a significant effect on tumor growth of the irradiated tumor nor on tumor progression of the abscopal tumor after RIT with anti-CTLA4. Furthermore, DAT and ICA did not significantly impact on the AE after RIT with anti-CTLA4 or anti-PD1. Surprisingly, ICA even appears to reduce outcomes after RIT with anti-CTLA4. Conclusion: We did not find a significant impact of combinatory ABx on the AE. Experimental application of the IFN-I-inducing metabolites DAT or ICA did not boost the AE after combined RIT. Additional studies are important to further investigate whether the intestinal microbiota or specific microbiota-derived metabolites modulate the AE.
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PURPOSE: Preoperative (neoadjuvant) radiation therapy (RT) is an essential part of multimodal rectal cancer therapy. Recently, total neoadjuvant therapy (TNT), which combines simultaneous radiochemotherapy with additional courses of chemotherapy, has emerged as an effective approach. TNT achieves a pathologic complete remission in approximately 30% of resected patients, opening avenues for treatment strategies that avoid radical organ resection. Furthermore, recent studies have demonstrated that anti-programmed cell death protein 1 immunotherapy can induce clinical complete responses in patients with specific genetic alterations. There is significant potential to enhance outcomes through intensifying, personalizing, and de-escalating treatment approaches. However, the heterogeneous response rates to RT or TNT and strategies to sensitize patients without specific genetic changes to immunotherapy remain poorly understood. METHODS AND MATERIALS: We developed a novel orthotopic mouse model of rectal cancer based on precisely defined endoscopic injections of tumor organoids that reflect tumor heterogeneity. Subsequently, we employed endoscopic- and computed tomography-guided RT and validated rectal tumor growth and response rates to therapy using small-animal magnetic resonance imaging and endoscopic follow-up. RESULTS: Rectal tumor formation was successfully induced in all mice after 2 organoid injections. Clinically relevant RT regimens with 5 × 5 Gy significantly delayed clinical signs of tumor progression and significantly improved survival. Consistent with human disease, rectal tumor progression correlated with the development of liver and lung metastases. Notably, long-term survivors after RT showed no evidence of tumor recurrence, as demonstrated by in vivo radiologic tumor staging and histopathologic examination. CONCLUSIONS: Our novel mouse model combines orthotopic tumor growth via noninvasive and precise rectal organoid injection and small-animal RT. This model holds significant promise for investigating the effect of tumor cell-intrinsic aspects, genetic alterations of the host, and exogenous factors (eg, nutrition or microbiota) on RT outcomes. Furthermore, it allows for the exploration of combination therapies involving chemotherapy, immunotherapy, or novel targeted therapies.
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Radioterapia Guiada por Imagem , Neoplasias Retais , Humanos , Animais , Camundongos , Recidiva Local de Neoplasia/patologia , Neoplasias Retais/patologia , Terapia Combinada , Quimiorradioterapia , Terapia Neoadjuvante , Estadiamento de NeoplasiasRESUMO
PURPOSE: The identification of internal mammary lymph node metastases and the assessment of associated risk factors are crucial for adjuvant regional lymph node irradiation in patients with breast cancer. The current study aims to investigate whether tumor contact with internal mammary perforator vessels is associated with gross internal mammary lymph node involvement. METHODS AND MATERIALS: We included 297 patients with primary breast cancer and gross internal mammary (IMN+) and/or axillary metastases as well as 230 patients without lymph node metastases. Based on pretreatment dynamic contrast-enhanced magnetic resonance imaging, we assessed contact of the tumor with the internal mammary perforating vessels (IMPV). RESULTS: A total of 59 patients had ipsilateral IMN+ (iIMN+), 10 patients had contralateral IMN+ (cIMN+), and 228 patients had ipsilateral axillary metastases without IMN; 230 patients had node-negative breast cancer. In patients with iIMN+, 100% of tumors had contact with ipsilateral IMPV, with 94.9% (n = 56) classified as major contact. In iIMN- patients, major IMPV contact was observed in only 25.3% (n = 116), and 36.2% (n = 166) had no IMPV contact at all. Receiver operating characteristic analysis revealed that "major IMPV contact" was more accurate in predicting iIMN+ (area under the curve, 0.85) compared with a multivariate model combining grade of differentiation, tumor site, size, and molecular subtype (area under the curve, 0.65). Strikingly, among patients with cIMN+, 100% of tumors had contact with a crossing contralateral IMPV, whereas in cIMN- patients, IMPVs to the contralateral side were observed in only 53.4% (iIMN+) and 24.8% (iIMN-), respectively. CONCLUSIONS: Tumor contact with the IMPV is highly associated with risk of gross IMN involvement. Further studies are warranted to investigate whether this identified risk factor is also associated with microscopic IMN involvement and whether it can assist in the selection of patients with breast cancer for irradiation of the internal mammary lymph nodes.
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Axila , Neoplasias da Mama , Linfonodos , Metástase Linfática , Imageamento por Ressonância Magnética , Humanos , Feminino , Pessoa de Meia-Idade , Neoplasias da Mama/patologia , Neoplasias da Mama/radioterapia , Fatores de Risco , Idoso , Adulto , Linfonodos/patologia , Linfonodos/diagnóstico por imagem , Idoso de 80 Anos ou mais , Artéria Torácica Interna/diagnóstico por imagemRESUMO
The microbiome is a predictor of clinical outcome in patients receiving allogeneic hematopoietic stem cell transplantation (allo-SCT). Microbiota-derived metabolites can modulate these outcomes. How bacteria, fungi and viruses contribute to the production of intestinal metabolites is still unclear. We combined amplicon sequencing, viral metagenomics and targeted metabolomics from stool samples of patients receiving allo-SCT (n = 78) and uncovered a microbiome signature of Lachnospiraceae and Oscillospiraceae and their associated bacteriophages, correlating with the production of immunomodulatory metabolites (IMMs). Moreover, we established the IMM risk index (IMM-RI), which was associated with improved survival and reduced relapse. A high abundance of short-chain fatty acid-biosynthesis pathways, specifically butyric acid via butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT, which catalyzes EC 2.8.3.8) was detected in IMM-RI low-risk patients, and virome genome assembly identified two bacteriophages encoding BCoAT as an auxiliary metabolic gene. In conclusion, our study identifies a microbiome signature associated with protective IMMs and provides a rationale for considering metabolite-producing consortia and metabolite formulations as microbiome-based therapies.
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Bacteriófagos , Transplante de Células-Tronco Hematopoéticas , Humanos , Bacteriófagos/genética , Fezes/microbiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Bactérias/genética , Bactérias/metabolismo , Ácido Butírico/metabolismoRESUMO
The results of this survey reveal current clinical practice in the handling of combined radioimmunotherapy with Immune Checkpoint Inhibitors (RT + ICI). We aim to provide a basis to open a discussion for clinical application of RT + ICI by analyzation of experts' assessment. We conducted a survey with 24 items with a focus on side effects of RT + ICI, common practice of scheduling and handling of adverse events. After pilot testing by radiation oncology experts the link to the online survey was sent to all members of the German Society of Radiation Oncology (DEGRO). In total, 51 radiation oncologists completed the questionnaire. Pulmonary toxicity under RT + ICI with ICIs was reported most frequently. Consensus was observed for bone and soft tissue RT of the limbs in favor for no interruption of ICIs. For cranial RT half of the participants do not suspend ICIs during normofractionated radiotherapy (nfRT) or stereotactic hypofractionated RT (SRT). More participants pause ICIs for central than for peripheral thoracic region. Maintenance therapy with ICIs is mostly not interrupted prior to RT. For management of RT associated pneumonitis under durvalumab the majority of 86.3% suggest corticosteroid therapy and 76.5% would postpone the next cycle of ICI therapy. The here obtained assessment and experiences by radiation oncologists reveal a large variability in practical handling of combined RT + ICI. Until scientific evidence is available a discussion for current clinical application of RT + ICI should be triggered. Interdisciplinary consensus guidelines with practical recommendations are required.
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Atitude do Pessoal de Saúde/etnologia , Radio-Oncologistas/psicologia , Radioimunoterapia/métodos , Adulto , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Competência Clínica , Feminino , Alemanha , Pessoal de Saúde/psicologia , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Radioimunoterapia/efeitos adversos , Radioimunoterapia/tendências , Estudos Retrospectivos , Inquéritos e QuestionáriosRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers. Innovative treatment concepts may enhance oncological outcome. Clinically relevant tumor models are essential in developing new therapeutic strategies. In the present study, we used two human PDAC cell lines for an orthotopic xenograft mouse model and compared treatment characteristics between this in vivo tumor model and PDAC patients. Tumor-bearing mice received stereotactic high-precision irradiation using arc technique after 3D-treatment planning. Induction of DNA damage in tumors and organs at risk (OARs) was histopathologically analyzed by the DNA damage marker γH2AX and compared with results after unprecise whole-abdomen irradiation. Our mouse model and preclinical setup reflect the characteristics of PDAC patients and clinical RT. It was feasible to perform stereotactic high-precision RT after defining tumor and OARs by CT imaging. After stereotactic RT, a high rate of DNA damage was mainly observed in the tumor but not in OARs. The calculated dose distributions and the extent of the irradiation field correlate with histopathological staining and the clinical example. We established and validated 3D-planned stereotactic RT in an orthotopic PDAC mouse model, which reflects the human RT. The efficacy of the whole workflow of imaging, treatment planning, and high-precision RT was proven by longitudinal analysis showing a significant improved survival. Importantly, this model can be used to analyze tumor regression and therapy-related toxicity in one model and will allow drawing clinically relevant conclusions.
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BACKGROUND: Antibody-mediated targeting of regulatory T cell receptors such as CTLA-4 enhances antitumor immune responses against several cancer entities including malignant melanoma. Yet, therapeutic success in patients remains variable underscoring the need for novel combinatorial approaches. METHODS: Here we established a vaccination strategy that combines engagement of the nucleic acid-sensing pattern recognition receptor RIG-I, antigen and CTLA-4 blockade. We used in vitro transcribed 5'-triphosphorylated RNA (3pRNA) to therapeutically target the RIG-I pathway. We performed in vitro functional analysis in bone-marrow derived dendritic cells and investigated RIG-I-enhanced vaccines in different murine melanoma models. FINDINGS: We found that protein vaccination together with RIG-I ligation via 3pRNA strongly synergizes with CTLA-4 blockade to induce expansion and activation of antigen-specific CD8+ T cells that translates into potent antitumor immunity. RIG-I-induced cross-priming of cytotoxic T cells as well as antitumor immunity were dependent on the host adapter protein MAVS and type I interferon (IFN-I) signaling and were mediated by dendritic cells. INTERPRETATION: Overall, our data demonstrate the potency of a novel combinatorial vaccination strategy combining RIG-I-driven immunization with CTLA-4 blockade to prevent and treat experimental melanoma. FUND: German Research Foundation (SFB 1335, SFB 1371), EMBO, Else Kröner-Fresenius-Foundation, German Cancer Aid, European Hematology Association, DKMS Foundation for Giving Life, Dres. Carl Maximilian and Carl Manfred Bayer-Foundation.
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Antígeno CTLA-4/imunologia , Vacinas Anticâncer/imunologia , Proteína DEAD-box 58/imunologia , Imunoterapia/métodos , Melanoma Experimental/terapia , RNA/imunologia , Adjuvantes Imunológicos/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Apresentação Cruzada , Proteína DEAD-box 58/genética , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
PURPOSE: Type I interferon (IFN-I) and interleukin (IL)-22 modulate regeneration of the thymus and intestinal epithelial cells (IECs) after cytotoxic stress such as irradiation. Radiation-induced damage to thymic tissues and IECs is a crucial aspect during the pathogenesis of inadequate immune reconstitution and acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with myeloablative total body irradiation (TBI), respectively. IL-22 and IFN-I reduce the severity of acute GVHD after allo-HSCT with myeloablative TBI. However, the role of biologically related type III interferon (IFN-III), also known as interferon lambda (IFN-λ) or IL-28, in this context is unclear. We therefore studied the role of the IFN-III pathway in thymic regeneration and GVHD after TBI and allo-HSCT. METHODS AND MATERIALS: Cohoused wild-type (WT) and IFN-III receptor-deficient (IL-28 receptor alpha subunit-deficient/IL-28Ra-/-) mice were analyzed in models of TBI-induced thymus damage and a model of GVHD after allo-HSCT with myeloablative TBI. PASylated IFN-III (PASylated IL-28A, XL-protein GmbH) was generated to prolong the plasma half-life of IFN-III. Pharmacologic activity and the effects of PASylated IL-28A on radiation-induced thymus damage and the course of GVHD after allo-HSCT with myeloablative TBI were tested. RESULTS: The course and severity of GVHD after myeloablative TBI and allo-HSCT in IL-28Ra-/- mice was comparable to those in WT mice. Activation of the IFN-III pathway by PASylated IL-28A did not significantly modulate GVHD after allo-HSCT with TBI. Furthermore, IL28Ra-/- mice and WT mice showed similar thymus regeneration after radiation, which could also not be significantly modulated by IFN-III receptor engagement using PASylated IL-28A. CONCLUSIONS: We analyzed the role of IFN-III signaling during radiation-mediated acute tissue injury. Despite molecular and biologic homologies with IFN-I and IL-22, IFN-III signaling did not improve thymus regeneration after radiation or the course of GVHD after myeloablative TBI and allo-HSCT.
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Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Interferons/metabolismo , Regeneração/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Timo/lesões , Timo/efeitos da radiação , Irradiação Corporal Total/efeitos adversos , Animais , Mucosa Intestinal/patologia , Mucosa Intestinal/efeitos da radiação , Intestinos/citologia , Camundongos , Timo/patologia , Timo/fisiopatologia , Interferon lambdaRESUMO
Resistance to cell death and evasion of immunosurveillance are major causes of cancer persistence and progression. Tumor cell-intrinsic activation of the RNA receptor retinoic acid-inducible gene-I (RIG-I) can trigger an immunogenic form of programmed tumor cell death, but its impact on antitumor responses remains largely unexplored. We show that activation of intrinsic RIG-I signaling induces melanoma cell death that enforces cross-presentation of tumor-associated antigens by bystander dendritic cells. This results in systemic expansion and activation of tumor-antigen specific T cells in vivo with subsequent regression of pre-established melanoma. These processes were dependent on the signaling hub MAVS and type I interferon (IFN-I) signaling in the host cell. Using melanoma cells deficient for the transcription factors IRF3 and IRF7, we demonstrate that RIG-I-activated tumor cells used as a vaccine are a relevant source of IFN-I during T cell cross-priming in vivo. Thus, our findings may facilitate translational development of personalized anticancer vaccines.
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Recent studies highlight immunoregulatory functions of type I interferons (IFN-I) during the pathogenesis of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We demonstrated that selective activation of IFN-I pathways including RIG-I/MAVS and cGAS/STING prior to allo-HSCT conditioning therapy can ameliorate the course of GVHD. However, direct effects of IFN-Is on immune cells remain ill characterized. We applied RIG-I agonists (3pRNA) to stimulate IFN-I production in murine models of conditioning therapy with total body irradiation (TBI) and GVHD. Using IFN-I receptor-deficient donor T cells and hematopoietic cells, we found that endogenous and RIG-I-induced IFN-Is do not reduce GVHD by acting on these cell types. However, 3pRNA applied before conditioning therapy reduced the ability of CD11c+ recipient cells to stimulate proliferation and interferon gamma expression of allogeneic T cells. Consistently, RIG-I activation before TBI reduced the proliferation of transplanted allogeneic T-cells. The reduced allogenicity of CD11c+ recipient cells was dependent on IFN-I signaling. Notably, this immunosuppressive function of DCs was restricted to a scenario where tissue damage occurs. Our findings uncover a context (damage by TBI) and IFN-I dependent modulation of T cells by DCs and extend the understanding about the cellular targets of IFN-I during allo-HSCT and GVHD.
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Células-Tronco Hematopoéticas/metabolismo , Interferon Tipo I/metabolismo , Ativação Linfocitária , Transdução de Sinais , Linfócitos T/citologia , Animais , Transplante de Medula Óssea , Antígeno CD11c/metabolismo , Morte Celular , Proliferação de Células , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Terapia de Imunossupressão , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Condicionamento Pré-Transplante , Transplante HomólogoRESUMO
Achieving durable clinical responses to immune checkpoint inhibitors remains a challenge. Here, we demonstrate that immunotherapy with anti-CTLA-4 and its combination with anti-PD-1 rely on tumor cell-intrinsic activation of the cytosolic RNA receptor RIG-I. Mechanistically, tumor cell-intrinsic RIG-I signaling induced caspase-3-mediated tumor cell death, cross-presentation of tumor-associated antigen by CD103+ dendritic cells, subsequent expansion of tumor antigen-specific CD8+ T cells, and their accumulation within the tumor tissue. Consistently, therapeutic targeting of RIG-I with 5'- triphosphorylated RNA in both tumor and nonmalignant host cells potently augmented the efficacy of CTLA-4 checkpoint blockade in several preclinical cancer models. In humans, transcriptome analysis of primary melanoma samples revealed a strong association between high expression of DDX58 (the gene encoding RIG-I), T cell receptor and antigen presentation pathway activity, and prolonged overall survival. Moreover, in patients with melanoma treated with anti-CTLA-4 checkpoint blockade, high DDX58 RIG-I transcriptional activity significantly associated with durable clinical responses. Our data thus identify activation of RIG-I signaling in tumors and their microenvironment as a crucial component for checkpoint inhibitor-mediated immunotherapy of cancer.
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Proteína DEAD-box 58/imunologia , Melanoma/imunologia , Animais , Linhagem Celular Tumoral , Estudos de Coortes , Proteína DEAD-box 58/genética , Modelos Animais de Doenças , Humanos , Imunoterapia , Melanoma/patologia , Melanoma/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microambiente TumoralRESUMO
The molecular pathways that regulate the tissue repair function of type I interferon (IFN-I) during acute tissue damage are poorly understood. We describe a protective role for IFN-I and the RIG-I/MAVS signaling pathway during acute tissue damage in mice. Mice lacking mitochondrial antiviral-signaling protein (MAVS) were more sensitive to total body irradiation- and chemotherapy-induced intestinal barrier damage. These mice developed worse graft-versus-host disease (GVHD) in a preclinical model of allogeneic hematopoietic stem cell transplantation (allo-HSCT) than did wild-type mice. This phenotype was not associated with changes in the intestinal microbiota but was associated with reduced gut epithelial integrity. Conversely, targeted activation of the RIG-I pathway during tissue injury promoted gut barrier integrity and reduced GVHD. Recombinant IFN-I or IFN-I expression induced by RIG-I promoted growth of intestinal organoids in vitro and production of the antimicrobial peptide regenerating islet-derived protein 3 γ (RegIIIγ). Our findings were not confined to RIG-I/MAVS signaling because targeted engagement of the STING (stimulator of interferon genes) pathway also protected gut barrier function and reduced GVHD. Consistent with this, STING-deficient mice suffered worse GVHD after allo-HSCT than did wild-type mice. Overall, our data suggest that activation of either RIG-I/MAVS or STING pathways during acute intestinal tissue injury in mice resulted in IFN-I signaling that maintained gut epithelial barrier integrity and reduced GVHD severity. Targeting these pathways may help to prevent acute intestinal injury and GVHD during allogeneic transplantation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína DEAD-box 58/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína DEAD-box 58/genética , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/metabolismo , Transplante de Células-Tronco Hematopoéticas , Interferon Tipo I/metabolismo , Intestinos/efeitos da radiação , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/fisiologia , Organoides/citologia , Organoides/metabolismo , Reação em Cadeia da Polimerase , Transdução de Sinais/fisiologia , Transplante HomólogoRESUMO
The success of allogeneic hematopoietic cell transplantation is limited by acute graft-versus-host disease (GvHD), a severe complication accompanied by high mortality rates. Yet, the molecular mechanisms initiating this disease remain poorly defined. In this study, we show that, after conditioning therapy, intestinal commensal bacteria and the damage-associated molecular pattern uric acid contribute to Nlrp3 inflammasome-mediated IL-1ß production and that gastrointestinal decontamination and uric acid depletion reduced GvHD severity. Early blockade of IL-1ß or genetic deficiency of the IL-1 receptor in dendritic cells (DCs) and T cells improved survival. The Nlrp3 inflammasome components Nlrp3 and Asc, which are required for pro-IL-1ß cleavage, were critical for the full manifestation of GvHD. In transplanted mice, IL-1ß originated from multiple intestinal cell compartments and exerted its effects on DCs and T cells, the latter being preferentially skewed toward Th17. Compatible with these mouse data, increased levels of active caspase-1 and IL-1ß were found in circulating leukocytes and intestinal GvHD lesions of patients. Thus, the identification of a crucial role for the Nlrp3 inflammasome sheds new light on the pathogenesis of GvHD and opens a potential new avenue for the targeted therapy of this severe complication.