RESUMEN
CD8+ T cells and natural killer (NK) cells are central cellular components of immune responses against pathogens and cancer, which rely on interleukin (IL)-15 for homeostasis. Here we show that IL-15 also mediates homeostatic priming of CD8+ T cells for antigen-stimulated activation, which is controlled by a deubiquitinase, Otub1. IL-15 mediates membrane recruitment of Otub1, which inhibits ubiquitin-dependent activation of AKT, a kinase that is pivotal for T cell activation and metabolism. Otub1 deficiency in mice causes aberrant responses of CD8+ T cells to IL-15, rendering naive CD8+ T cells hypersensitive to antigen stimulation characterized by enhanced metabolic reprograming and effector functions. Otub1 also controls the maturation and activation of NK cells. Deletion of Otub1 profoundly enhances anticancer immunity by unleashing the activity of CD8+ T cells and NK cells. These findings suggest that Otub1 controls the activation of CD8+ T cells and NK cells by functioning as a checkpoint of IL-15-mediated priming.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Cisteína Endopeptidasas/metabolismo , Interleucina-15/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Cisteína Endopeptidasas/deficiencia , Enzimas Desubicuitinizantes/metabolismo , Modelos Animales de Enfermedad , Metabolismo Energético , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Interleucina-15/genética , Melanoma Experimental , Ratones , Ratones Transgénicos , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Interleucina-15/metabolismo , Autotolerancia/genética , Autotolerancia/inmunología , Transducción de Señal , Especificidad del Receptor de Antígeno de Linfocitos T , UbiquitinaciónRESUMEN
Coeliac disease is a complex, polygenic inflammatory enteropathy caused by exposure to dietary gluten that occurs in a subset of genetically susceptible individuals who express either the HLA-DQ8 or HLA-DQ2 haplotypes1,2. The need to develop non-dietary treatments is now widely recognized3, but no pathophysiologically relevant gluten- and HLA-dependent preclinical model exists. Furthermore, although studies in humans have led to major advances in our understanding of the pathogenesis of coeliac disease4, the respective roles of disease-predisposing HLA molecules, and of adaptive and innate immunity in the development of tissue damage, have not been directly demonstrated. Here we describe a mouse model that reproduces the overexpression of interleukin-15 (IL-15) in the gut epithelium and lamina propria that is characteristic of active coeliac disease, expresses the predisposing HLA-DQ8 molecule, and develops villous atrophy after ingestion of gluten. Overexpression of IL-15 in both the epithelium and the lamina propria is required for the development of villous atrophy, which demonstrates the location-dependent central role of IL-15 in the pathogenesis of coeliac disease. In addition, CD4+ T cells and HLA-DQ8 have a crucial role in the licensing of cytotoxic T cells to mediate intestinal epithelial cell lysis. We also demonstrate a role for the cytokine interferon-γ (IFNγ) and the enzyme transglutaminase 2 (TG2) in tissue destruction. By reflecting the complex interaction between gluten, genetics and IL-15-driven tissue inflammation, this mouse model provides the opportunity to both increase our understanding of coeliac disease, and develop new therapeutic strategies.
Asunto(s)
Enfermedad Celíaca/inmunología , Enfermedad Celíaca/patología , Glútenes/inmunología , Antígenos HLA-DQ/inmunología , Interleucina-15/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Femenino , Antígenos HLA-DQ/genética , Humanos , Interferón gamma/inmunología , Interleucina-15/genética , Masculino , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismoRESUMEN
Tissue-resident memory T cells (T(RM) cells) provide superior protection against infection in extralymphoid tissues. Here we found that CD103(+)CD8(+) T(RM) cells developed in the skin from epithelium-infiltrating precursor cells that lacked expression of the effector-cell marker KLRG1. A combination of entry into the epithelium plus local signaling by interleukin 15 (IL-15) and transforming growth factor-ß (TGF-ß) was required for the formation of these long-lived memory cells. Notably, differentiation into T(RM) cells resulted in the progressive acquisition of a unique transcriptional profile that differed from that of circulating memory cells and other types of T cells that permanently reside in skin epithelium. We provide a comprehensive molecular framework for the local differentiation of a distinct peripheral population of memory cells that forms a first-line immunological defense system in barrier tissues.
Asunto(s)
Antígenos CD/inmunología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Cadenas alfa de Integrinas/inmunología , Transducción de Señal/inmunología , Piel/inmunología , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Citometría de Flujo , Herpes Simple/inmunología , Herpes Simple/virología , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno/inmunología , Cadenas alfa de Integrinas/genética , Cadenas alfa de Integrinas/metabolismo , Interleucina-15/genética , Interleucina-15/inmunología , Interleucina-15/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Piel/metabolismo , Piel/virología , Transcriptoma/genética , Transcriptoma/inmunologíaRESUMEN
Despite the remarkable success of chimeric antigen receptor (CAR) T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous interferon (IFN)-γ promoter for transgenic interleukin (IL)-15 expression. We demonstrate that IFN-γ expression is tightly regulated by T cell receptor signaling. By introducing an internal ribosome entry site IL15 into the 3' UTR of the IFN-γ gene via homology directed repair-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Last, as a part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.
Asunto(s)
Inmunoterapia Adoptiva , Interferón gamma , Interleucina-15 , Regiones Promotoras Genéticas , Receptores Quiméricos de Antígenos , Interferón gamma/metabolismo , Humanos , Animales , Ratones , Inmunoterapia Adoptiva/métodos , Interleucina-15/genética , Interleucina-15/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/metabolismo , Linfocitos T/inmunología , Vectores Genéticos/genética , Línea Celular Tumoral , Transgenes , Citocinas/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Expresión GénicaRESUMEN
The development of innate lymphoid cell (ILC) transcription factor reporter mice has shown a previously unexpected complexity in ILC hematopoiesis. Using novel polychromic mice to achieve higher phenotypic resolution, we have characterized bone marrow progenitors that are committed to the group 1 ILC lineage. These common ILC1/NK cell progenitors (ILC1/NKP), which we call "aceNKPs", are defined as lineage-Id2+IL-7Rα+CD25-α4ß7-NKG2A/C/E+Bcl11b-. In vitro, aceNKPs differentiate into group 1 ILCs, including NK-like cells that express Eomes without the requirement for IL-15, and produce IFN-γ and perforin upon IL-15 stimulation. Following reconstitution of Rag2-/-Il2rg-/- hosts, aceNKPs give rise to a spectrum of mature ILC1/NK cells (regardless of their tissue location) that cannot be clearly segregated into the traditional ILC1 and NK subsets, suggesting that group 1 ILCs constitute a dynamic continuum of ILCs that can develop from a common progenitor. In addition, aceNKP-derived ILC1/NK cells effectively ameliorate tumor burden in a model of lung metastasis, where they acquired a cytotoxic NK cell phenotype. Our results identify the primary ILC1/NK progenitor that lacks ILC2 or ILC3 potential and is strictly committed to ILC1/NK cell production irrespective of tissue homing.
Asunto(s)
Inmunidad Innata , Interleucina-15 , Animales , Ratones , Interleucina-15/genética , Células Asesinas Naturales , Perforina , Factores de Transcripción , Proteínas Represoras , Proteínas Supresoras de TumorRESUMEN
This study aimed to evaluate the role of skeletal muscle-derived interleukin (IL)-15 in the regulation of skeletal muscle autophagy using IL-15 knockout (KO) and transgenic (TG) mice. Male C57BL/6 wild-type (WT), IL-15 KO, and IL-15 TG mice were used in this study. Changes in muscle mass, forelimb grip strength, succinate dehydrogenase (SDH) activity, gene and protein expression levels of major regulators and indicators of autophagy, comprehensive gene expression, and DNA methylation in the gastrocnemius muscle were analyzed. Enrichment pathway analyses revealed that the pathology of IL-15 gene deficiency was related to the autophagosome pathway. Moreover, although IL-15 KO mice maintained gastrocnemius muscle mass, they exhibited a decrease in autophagy induction. IL-15 TG mice exhibited a decrease in gastrocnemius muscle mass and an increase in forelimb grip strength and SDH activity in skeletal muscle. In the gastrocnemius muscle, the ratio of phosphorylated adenosine monophosphate-activated protein kinase α (AMPKα) to total AMPKα and unc-51-like autophagy activating kinase 1 and Beclin1 protein expression were higher in the IL-15 TG group than in the WT group. IL-15 gene deficiency induces a decrease in autophagy induction. In contrast, IL-15 overexpression could improve muscle quality by activating autophagy induction while decreasing muscle mass. The regulation of IL-15 in autophagy in skeletal muscles may lead to the development of therapies for the autophagy-induced regulation of skeletal muscle mass and cellular quality control.NEW & NOTEWORTHY IL-15 gene deficiency can decrease autophagy induction. However, although IL-15 overexpression induced a decrease in muscle mass, it led to an improvement in muscle quality. Based on these results, understanding the role of IL-15 in regulating autophagy pathways within skeletal muscle may lead to the development of therapies for the autophagy-induced regulation of skeletal muscle mass and cellular quality control.
Asunto(s)
Interleucina-15 , Músculo Esquelético , Ratones , Masculino , Animales , Interleucina-15/genética , Interleucina-15/metabolismo , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Ratones Transgénicos , Ratones Noqueados , Proteínas Quinasas Activadas por AMP/metabolismo , AutofagiaRESUMEN
BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has shown remarkable responses in hematological malignancies with several approved products, but not in solid tumors. Patients suffer from limited response and tumor relapse due to low efficacy of CAR-T cells in the complicated and immunosuppressive tumor microenvironment. This clinical challenge has called for better CAR designs and combined strategies to improve CAR-T cell therapy against tumor changes. METHODS: In this study, IL-15/IL-15Rα was inserted into the extracellular region of CAR targeting mesothelin. In-vitro cytotoxicity and cytokine production were detected by bioluminescence-based killing and ELISA respectively. In-vivo xenograft mice model was used to evaluate the anti-tumor effect of CAR-T cells. RNA-sequencing and online database analysis were used to identify new targets in residual gastric cancer cells after cytotoxicity assay. CAR-T cell functions were detected in vitro and in vivo after GLI Pathogenesis Related 1 (GLIPR1) knockdown in gastric cancer cells. Cell proliferation and migration of gastric cancer cells were detected by CCK-8 and scratch assay respectively after GLIPR1 were overexpressed or down-regulated. RESULTS: CAR-T cells constructed with IL-15/IL-15Rα (CAR-ss-T) showed significantly improved CAR-T cell expansion, cytokine production and cytotoxicity, and resulted in superior tumor control compared to conventional CAR-T cells in gastric cancer. GLIPR1 was up-regulated after CAR-T treatment and survival was decreased in gastric cancer patients with high GLIPR1 expression. Overexpression of GLIPR1 inhibited cytotoxicity of conventional CAR-T but not CAR-ss-T cells. CAR-T treatment combined with GLIPR1 knockdown increased anti-tumor efficacy in vitro and in vivo. CONCLUSIONS: Our data demonstrated for the first time that this CAR structure design combined with GLIPR1 knockdown in gastric cancer improved CAR-T cell-mediated anti-tumor response.
Asunto(s)
Receptores Quiméricos de Antígenos , Neoplasias Gástricas , Humanos , Animales , Ratones , Neoplasias Gástricas/genética , Neoplasias Gástricas/terapia , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Interleucina-15/genética , Interleucina-15/metabolismo , Línea Celular Tumoral , Recurrencia Local de Neoplasia/metabolismo , Inmunoterapia Adoptiva/métodos , Linfocitos T , Ensayos Antitumor por Modelo de Xenoinjerto , Microambiente Tumoral , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismoRESUMEN
The present study investigated the relationship between single nucleotide polymorphisms in the interleukin (IL)-15 gene located (exon 8) on the chromosomal location 4q31.21 and acute lymphoblastic leukaemia (ALL) risk in Iraqi patients. A total of 78 (49 male -29 female) primary ALL (62B-cell, 16 T-cells lineages cases and 30 healthy control subjects (median age 11, age range were 4-21.5), were enrolled at the Nanakaly Hospital of Erbil Province between February 2021 and January 2022. The genotype analysis was performed using polymerase chain reaction (PCR) and Sanger DNA sequencing. The IL15 homozygous rs10833 (100%) and rs2291596 (63.6%) genotypes indicated high frequencies and were associated with a risk of developing ALL, while the remaining 16 novel mutations indicated in low frequency (9.1%) except for the 97270G>GT genotype (18.2%). High expression levels were noted for different clusters of differentiation (CD) biomarkers between both subtypes of ALL, including, CD10, CD19, CD22, CD79a, CD99, terminal deoxynucleotidyl transferase (TdT), and human leukocyte antigen DR (HLA-DR) isotype in B-cells lineages, while, CD2, CD3, CD5, CD7, CD13, CD117 and TdT are more specific to T-cells lineages. On the other hand, significant changes were noted in certain hematological parameters including red blood cells (RBCs), haemoglobin (g/dl), haematocrite (HCt %), red blood cell distribution width (RDW %), and platelet counts (PLT- 109/L) compared with those of healthy subjects. Finally, it was concluded that various novel mutations were recorded with different subtypes of ALL diseases, and mild anemia was observed among patients. Future studies will be towered to associate these mutations with prognosis and therapeutic response of diseases.
Asunto(s)
Predisposición Genética a la Enfermedad , Interleucina-15 , Mutación , Polimorfismo de Nucleótido Simple , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adolescente , Niño , Preescolar , Femenino , Humanos , Masculino , Adulto Joven , Estudios de Casos y Controles , Frecuencia de los Genes/genética , Genotipo , Interleucina-15/genética , Mutación/genética , Polimorfismo de Nucleótido Simple/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Factores de RiesgoRESUMEN
Autosomal recessive (AR) and dominant (AD) deficiencies of TLR3 and TRIF are believed to be crucial genetic causes of herpes simplex encephalitis (HSE), which is a fatal disease causing focal or global cerebral dysfunction following infection with herpes simplex virus type 1 (HSV-1). However, few studies have been conducted on the immunopathological networks of HSE in the context of TLR3 and TRIF defects at the cellular and molecular levels. In this work, we deciphered the crosstalk between type I IFN (IFN-I)-producing epithelial layer and IL-15-producing dendritic cells (DC) to activate NK cells for the protective role of TLR3/TRIF pathway in HSE progression after vaginal HSV-1 infection. TLR3- and TRIF-ablated mice showed enhanced susceptibility to HSE progression, along with high HSV-1 burden in vaginal tract, lymphoid tissues and CNS. The increased HSV-1 burden in TLR3- and TRIF-ablated mice did not correlate with increased infiltration of Ly-6C+ monocytes, but it was closely associated with impaired NK cell activation in vaginal tract. Furthermore, using delicate ex vivo experiments and bone marrow transplantation, TRIF deficiency in tissue-resident cells, such as epithelial cells in vaginal tract, was found to cause impaired NK cell activation by means of low IFN-I production, whereas IFN-I receptor in DC was required for NK cell activation via IL-15 production in response to IFN-I produced from epithelial layer. These results provide new information about IFN-I- and IL-15-mediated crosstalk between epithelial cells and DC at the primary infection site, which suppresses HSE progression in a TLR3- and TRIF-dependent manner.
Asunto(s)
Encefalitis por Herpes Simple , Herpesvirus Humano 1 , Femenino , Animales , Ratones , Encefalitis por Herpes Simple/genética , Receptor Toll-Like 3/genética , Interleucina-15/genética , Células Dendríticas , Proteínas Adaptadoras del Transporte Vesicular/genéticaRESUMEN
KD033 is a clinical-stage immunocytokine composed of a high-affinity anti-human-PD-L1 antibody and the human IL-15/ IL-15 receptor sushi-domain complex. We have previously shown that KD033-surrogate, the anti-mouse-PD-L1/IL-15 immunocytokine, was efficacious in several syngeneic murine tumor models including those that were refractory to anti-PD-1/PD-L1 checkpoint blockers. KD033-surrogate showed better efficacy than the combination treatment of its component, anti-PD-L1 antibody with the non-targeting IL-15. KD033-surrogate was also efficacious in both low and high PD-L1-expressing tumors. In this study, we have utilized double knock-in mice expressing functional human PD-1/PD-L1 to show that the clinical molecule, KD033, reproduced the anti-tumor efficacy observed with KD033-surrogate in the syngeneic models. KD033 was equally efficacious in reducing the growth of human-PD-L1 positive (hPDL1+) and negative (hPDL1-) MC38 murine tumors. We observed similar peripheral pharmacodynamics changes in KD033-treated mice bearing either hPDL1+ or hPDL1- MC38 tumors. However, different transcriptomic profiles were observed between KD033-treated hPDL1+ and hPDL1- MC38 tumors with marked changes involving mostly downregulated genes in hPDL1- tumors in addition to the immune-related genes changes observed in both hPDL1+ and hPDL1- MC38 tumors. Cytotoxic and myeloid cell signatures were upregulated in both tumors with relatively greater increases observed in hPDL1- MC38 tumors. These effects of KD033 treatment in PD-L1 positive and negative tumors demonstrate the role of PD-L1 in targeting of IL-15 cytokine in vivo.
Asunto(s)
Antineoplásicos , Neoplasias , Animales , Humanos , Ratones , Antineoplásicos/farmacología , Antígeno B7-H1/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Interleucina-15/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteínas Recombinantes de Fusión/farmacología , Transducción de SeñalRESUMEN
The IL-15 superagonist N-803 has been shown to enhance the function of CD8 T cells and NK cells. We previously found that in a subset of vaccinated, ART-naive, SIV+ rhesus macaques, N-803 treatment led to a rapid but transient decline in plasma viremia that positively correlated with an increase in the frequency of CD8 T cells. Here, we tested the hypothesis that prophylactic vaccination was required for the N-803 mediated suppression of SIV plasma viremia. We either vaccinated rhesus macaques with a DNA prime/Ad5 boost regimen using vectors expressing SIVmac239 gag with or without a plasmid expressing IL-12 or left them unvaccinated. The animals were then intravenously infected with SIVmac239M. 6 months after infection, the animals were treated with N-803. We found no differences in the control of plasma viremia during N-803 treatment between vaccinated and unvaccinated macaques. Interestingly, when we divided the SIV+ animals based on their plasma viral load set-points prior to the N-803 treatment, N-803 increased the frequency of SIV-specific T cells expressing ki-67+ and granzyme B+ in animals with low plasma viremia (<104 copies/mL; SIV controllers) compared to animals with high plasma viremia (>104 copies/mL; SIV noncontrollers). In addition, Gag-specific CD8 T cells from the SIV+ controllers had a greater increase in CD8+CD107a+ T cells in ex vivo functional assays than did the SIV+ noncontrollers. Overall, our results indicate that N-803 is most effective in SIV+ animals with a preexisting immunological ability to control SIV replication. IMPORTANCE N-803 is a drug that boosts the immune cells involved in combating HIV/SIV infection. Here, we found that in SIV+ rhesus macaques that were not on antiretroviral therapy, N-803 increased the proliferation and potential capacity for killing of the SIV-specific immune cells to a greater degree in animals that spontaneously controlled SIV than in animals that did not control SIV. Understanding the mechanism of how N-803 might function differently in individuals that control HIV/SIV (for example, individuals on antiretroviral therapy or spontaneous controllers) compared to settings where HIV/SIV are not controlled, could impact the efficacy of N-803 utilization in the field of HIV cure.
Asunto(s)
Infecciones por VIH , Síndrome de Inmunodeficiencia Adquirida del Simio , Virus de la Inmunodeficiencia de los Simios , Animales , Macaca mulatta , Interleucina-15/genética , Granzimas , Viremia , Antígeno Ki-67 , Linfocitos T CD8-positivos , Antirretrovirales/uso terapéutico , Carga Viral , Infecciones por VIH/tratamiento farmacológico , Interleucina-12 , ADNRESUMEN
Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2-containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation "armored" chimeric antigen receptor (CAR) engineering of cord blood-derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15-secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.
Asunto(s)
Sangre Fetal/citología , Inmunoterapia Adoptiva , Interleucina-15/genética , Células Asesinas Naturales/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Supresoras de la Señalización de Citocinas/antagonistas & inhibidores , Aerobiosis , Animales , Antígenos CD19/inmunología , Linfoma de Burkitt/patología , Linfoma de Burkitt/terapia , Sistemas CRISPR-Cas , Línea Celular Tumoral , Técnicas de Inactivación de Genes , Glucólisis , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Interleucina-15/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/trasplante , Diana Mecanicista del Complejo 1 de la Rapamicina/fisiología , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Receptores Quiméricos de Antígenos , Transducción de Señal/fisiología , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Human innate immunity plays a critical role in tumor surveillance and in immunoregulation within the tumor microenvironment. Natural killer (NK) cells are innate lymphoid cells that have opposing roles in the tumor microenvironment, including NK cell subsets that mediate tumor cell cytotoxicity and subsets with regulatory function that contribute to the tumor immune suppressive environment. The balance between effector and regulatory NK cell subsets has been studied extensively in murine models of cancer, but there is a paucity of models to study human NK cell function in tumorigenesis. Humanized mice are a powerful alternative to syngeneic mouse tumor models for the study of human immuno-oncology and have proven effective tools to test immunotherapies targeting T cells. However, human NK cell development and survival in humanized NOD-scid-IL2rgnull (NSG) mice are severely limited. To enhance NK cell development, we have developed NSG mice that constitutively expresses human Interleukin 15 (IL15), NSG-Tg(Hu-IL15). Following hematopoietic stem cell engraftment of NSG-Tg(Hu-IL15) mice, significantly higher levels of functional human CD56+ NK cells are detectable in blood and spleen, as compared to NSG mice. Hematopoietic stem cell (HSC)-engrafted NSG-Tg(Hu-IL15) mice also supported the development of human CD3+ T cells, CD20+ B cells, and CD33+ myeloid cells. Moreover, the growth kinetics of a patient-derived xenograft (PDX) melanoma were significantly delayed in HSC-engrafted NSG-Tg(Hu-IL15) mice as compared to HSC-engrafted NSG mice demonstrating that human NK cells have a key role in limiting the tumor growth. Together, these data demonstrate that HSC-engrafted NSG-Tg(Hu-IL15) mice support enhanced development of functional human NK cells, which limit the growth of PDX tumors.
Asunto(s)
Inmunidad Innata , Interleucina-15 , Animales , Modelos Animales de Enfermedad , Humanos , Subunidad gamma Común de Receptores de Interleucina/genética , Interleucina-15/genética , Células Asesinas Naturales , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCIDRESUMEN
CD8αα(+) intraepithelial lymphocytes (IELs) are instrumental in maintaining the epithelial barrier in the intestine. Similar to natural killer cells and other innate lymphoid cells, CD8αα(+) IELs constitutively express the T-box transcription factor T-bet. However, the precise role of T-bet for the differentiation or function of IELs is unknown. Here we show that mice genetically deficient for T-bet lacked both TCRαß(+) and TCRγδ(+) CD8αα(+) IELs and thus are more susceptible to chemically induced colitis. Although T-bet was induced in thymic IEL precursors (IELPs) as a result of agonist selection and interleukin-15 (IL-15) receptor signaling, it was dispensable for the generation of IELPs. Subsequently, T-bet was required for the IL-15-dependent activation, differentiation, and expansion of IELPs in the periphery. Our study reveals a function of T-bet as a central transcriptional regulator linking agonist selection and IL-15 signaling with the emergence of CD8αα(+) IELs.
Asunto(s)
Antígenos CD8/biosíntesis , Interleucina-15/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Proteínas de Dominio T Box/genética , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Colitis/inducido químicamente , Colitis/inmunología , Células Epiteliales/inmunología , Interleucina-15/inmunología , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Intestinos/citología , Intestinos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Interleucina-15/inmunología , Transducción de Señal/inmunología , Proteínas de Dominio T Box/biosíntesisRESUMEN
Acute myeloid leukemia (AML) is a heterogeneous malignancy characterized by the clonal expansion of myeloid precursor cells in the bone marrow. In this study, we investigated the interplay of hematological parameters, CD markers, genetic polymorphisms, and database mutations in the interleukin 15 (IL15) gene in AML patients. We enrolled 59 newly diagnosed AML patients and analyzed their bone marrow specimens using flow cytometry and molecular techniques. The hematological parameters of the AML patients revealed a significant increase in platelet count and RBC, Hb, and HCT levels compared to healthy individuals. CD marker expression analysis revealed upregulation of CD33, CD45, CD13, CD117, CD38, HLA-DR, CD15, CD64, MPO, CD34, and CD11c in AML patients. Molecular analysis showed 15 mutations in different positions of exon 8 of the IL15 gene, with the most frequent mutation being a homozygous mutation resulting from a nucleotide substitution. Additionally, 10 novel heterozygous mutations were identified in different locations of chromosome 4, with a low variant rate. Finally, database analysis of gnomAD and Mutagene revealed a high number of potential driver mutations in the IL15 gene in leukemia patients. These results provide valuable insights into the genetic and immunophenotypic characteristics of AML patients and highlight the potential role of IL15 in AML pathogenesis.
Asunto(s)
Interleucina-15 , Leucemia Mieloide Aguda , Humanos , Interleucina-15/genética , Antígenos CD/metabolismo , Leucemia Mieloide Aguda/genética , Mutación/genética , Inmunofenotipificación , Citometría de Flujo/métodos , Polimorfismo GenéticoRESUMEN
Preeclampsia (PE) is a pregnancy-associated complication accompanied by gestational hypertension and proteinuria, affecting 2-8% of pregnancies globally. The placental trophoblast cell invasion of decidua and myometrium during early gestation is crucial for healthy placentation. Thus, trophoblast dysfunction might contribute to PE onset. Therefore, further investigations are needed to elucidate the underlying mechanism of trophoblast cell functions. In the present study, we identified a novel pseudogene named C-Type Lectin Domain Family 4 Member G Pseudogene 1 (CLEC4GP1), which was aberrantly expressed in PE placental tissues. In vitro analyses showed that CLEC4GP1 overexpression significantly increased the cell viability and invasiveness and decreased the apoptosis rate of HTR-8/SVneo and JEG-3 cells, while CLEC4GP1 knockdown exerted opposite effects, suggesting the beneficial role of CLEC4GP1 in trophoblast cells. Next, co-expression analysis found that CLEC4GP1 was negatively correlated with Interleukin 15 (IL-15). The expression of IL-15 dramatically increased in PE placental tissues. In HTR-8/SVneo and JEG-3 cells, IL-15 exhibited detrimental effects, opposite to CLEC4GP1, and they were negatively correlated. In addition, CLEC4GP1 attenuates the mRNA stability of IL-16 by inhibiting the binding between human antigen R (HuR) protein and IL-15 RNA. Finally, the obverse effects of CLEC4GP1 and IL-15 were investigated, and results showed that IL-15 reverted CLEC4GP1 induced cellular functions. In brief, these data suggest that CLEC4GP1/IL-15 axis might modulate the occurrence and progression of PE via influencing the trophoblast cell viability, apoptosis, and invasive capability. This study provided cognizance of targeting the CLEC4GP1/IL-15 axis as a novel therapeutic approach to mitigate PE progression.
Asunto(s)
Preeclampsia , Trofoblastos , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Humanos , Interleucina-15/genética , Placenta/metabolismo , Preeclampsia/genética , Preeclampsia/metabolismo , Embarazo , Seudogenes/genética , Trofoblastos/metabolismoRESUMEN
Hepatic adeno-associated viral (AAV) gene transfer has the potential to cure the X-linked bleeding disorder hemophilia A. However, declining therapeutic coagulation factor VIII (FVIII) expression has plagued clinical trials. To assess the mechanistic underpinnings of this loss of FVIII expression, we developed a hemophilia A mouse model that shares key features observed in clinical trials. Following liver-directed AAV8 gene transfer in the presence of rapamycin, initial FVIII protein expression declines over time in the absence of antibody formation. Surprisingly, loss of FVIII protein production occurs despite persistence of transgene and mRNA, suggesting a translational shutdown rather than a loss of transduced hepatocytes. Some of the animals develop ER stress, which may be linked to hepatic inflammatory cytokine expression. FVIII protein expression is preserved by interleukin-15/interleukin-15 receptor blockade, which suppresses CD8+ T and natural killer cell responses. Interestingly, mice with initial FVIII levels >100% of normal had diminishing expression while still under immune suppression. Taken together, our findings of interanimal variability of the response, and the ability of the immune system to shut down transgene expression without utilizing cytolytic or antibody-mediated mechanisms, illustrate the challenges associated with FVIII gene transfer. Our protocols based upon cytokine blockade should help to maintain efficient FVIII expression.
Asunto(s)
Factor VIII , Interleucina-15 , Ratones , Animales , Factor VIII/genética , Interleucina-15/genética , Sirolimus/farmacologíaRESUMEN
Chimeric fusion transcription factors are oncogenic hallmarks of several devastating cancer entities including pediatric sarcomas, such as Ewing sarcoma (EwS) and alveolar rhabdomyosarcoma (ARMS). Despite their exquisite specificity, these driver oncogenes have been considered largely undruggable due to their lack of enzymatic activity.Here, we show in the EwS model that - capitalizing on neomorphic DNA-binding preferences - the addiction to the respective fusion transcription factor EWSR1-FLI1 can be leveraged to express therapeutic genes.We genetically engineered a de novo enhancer-based, synthetic and highly potent expression cassette that can elicit EWSR1-FLI1-dependent expression of a therapeutic payload as evidenced by episomal and CRISPR-edited genomic reporter assays. Combining in silico screens and immunohistochemistry, we identified GPR64 as a highly specific cell surface antigen for targeted transduction strategies in EwS. Functional experiments demonstrated that anti-GPR64-pseudotyped lentivirus harboring our expression cassette can specifically transduce EwS cells to promote the expression of viral thymidine kinase sensitizing EwS for treatment to otherwise relatively non-toxic (Val)ganciclovir and leading to strong anti-tumorigenic, but no adverse effects in vivo. Further, we prove that similar vector designs can be applied in PAX3-FOXO1-driven ARMS, and to express immunomodulatory cytokines, such as IL-15 and XCL1, in tumor entities typically considered to be immunologically 'cold'.Collectively, these results generated in pediatric sarcomas indicate that exploiting, rather than suppressing, the neomorphic functions of chimeric transcription factors may open inroads to innovative and personalized therapies, and that our highly versatile approach may be translatable to other cancers addicted to oncogenic transcription factors with unique DNA-binding properties.
Asunto(s)
Sarcoma de Ewing , Sarcoma , Antígenos de Superficie/uso terapéutico , Línea Celular Tumoral , Niño , ADN , Ganciclovir/uso terapéutico , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-15/genética , Interleucina-15/metabolismo , Interleucina-15/uso terapéutico , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Sarcoma/genética , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/terapia , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Timidina Quinasa/uso terapéuticoRESUMEN
Despite the success of antiretroviral therapy (ART) to halt viral replication and slow disease progression, this treatment is not curative and there remains an urgent need to develop approaches to clear the latent HIV reservoir. The human IL-15 superagonist N-803 (formerly ALT-803) is a promising anti-cancer biologic with potent immunostimulatory properties that has been extended into the field of HIV as a potential "shock and kill" therapeutic for HIV cure. However, the ability of N-803 to reactivate latent virus and modulate anti-viral immunity in vivo under the cover of ART remains undefined. Here, we show that in ART-suppressed, simian-human immunodeficiency virus (SHIV)SF162P3-infected rhesus macaques, subcutaneous administration of N-803 activates and mobilizes both NK cells and SHIV-specific CD8+ T cells from the peripheral blood to lymph node B cell follicles, a sanctuary site for latent virus that normally excludes such effector cells. We observed minimal activation of memory CD4+ T cells and no increase in viral RNA content in lymph node resident CD4+ T cells post N-803 administration. Accordingly, we found no difference in the number or magnitude of plasma viremia timepoints between treated and untreated animals during the N-803 administration period, and no difference in the size of the viral DNA cell-associated reservoir post N-803 treatment. These results substantiate N-803 as a potent immunotherapeutic candidate capable of activating and directing effector CD8+ T and NK cells to the B cell follicle during full ART suppression, and suggest N-803 must be paired with a bona fide latency reversing agent in vivo to facilitate immune-mediated modulation of the latent viral reservoir.
Asunto(s)
Antirretrovirales/administración & dosificación , Linfocitos B/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Interleucina-15/antagonistas & inhibidores , Células Asesinas Naturales/efectos de los fármacos , Proteínas/administración & dosificación , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/fisiopatología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Interleucina-15/genética , Interleucina-15/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Macaca mulatta , Proteínas Recombinantes de Fusión , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Virus de la Inmunodeficiencia de los Simios/fisiología , Latencia del Virus/efectos de los fármacosRESUMEN
Vulvovaginal candidiasis (VVC), a major gynecological disease with high recurrence rate, increases the risk of abortion, intrauterine infection, premature rupture of membranes, and premature birth in pregnancy. However, the exact pathogenesis of this disease has yet to be elucidated. To facilitate understanding of the pathogenesis of VVC in pregnancy, this study sought to establish an animal model of vaginal infection with Candida albicans in pregnant mice. Female mice were mated with male mice, and female mice were infected with C. albicans at E4.5 (embryonic day 4.5). The weight and abortion rate of pregnant mice at E0.5, E4.5, E8.5, E11.5, and E18.5 were recorded, respectively, as well as the weights of fetus and placenta on E18.5. Fetal weight at E18.5 and the weight growth rate in the experimental mice was lower than those in the control mice, but the placenta weight at E18.5 and the abortion rate in the experimental mice were increased with those of the control mice. Hematoxylin-eosin (H&E) staining, Gomori-Grocott staining and vaginal lavage culturing were conducted to verify that the experimental mice were infected with C. albicans. Differentially expressed gene IL-15 was screened out by polymerase chain reaction (PCR) array between the two groups. Enzyme-linked immunosorbent assay (ELISA) showed that IL-15 expression in plasma of the mice was decreased in the experimental group compared with the control group. RT-qPCR confirmed that IL-15 mRNA expression was increased in placental tissues, while mRNA expression of IL-15R/JAK1-JAK3/PI3K/PDK1/AKT/P70S6K-mTOR was decreased in placental tissues. In conclusion, this study demonstrated that VVC in BALB/c pregnant mice led to a series of adverse pregnancy outcomes that were related to changes in IL-15 and its downstream signaling pathways, which may indicate a potential therapy for VVC during pregnancy in humans.