RESUMEN
CD4+ T cells can either enhance or inhibit tumour immunity. Although regulatory T cells have long been known to impede antitumour responses1-5, other CD4+ T cells have recently been implicated in inhibiting this response6,7. Yet, the nature and function of the latter remain unclear. Here, using vaccines containing MHC class I (MHC-I) neoantigens (neoAgs) and different doses of tumour-derived MHC-II neoAgs, we discovered that whereas the inclusion of vaccines with low doses of MHC-II-restricted peptides (LDVax) promoted tumour rejection, vaccines containing high doses of the same MHC-II neoAgs (HDVax) inhibited rejection. Characterization of the inhibitory cells induced by HDVax identified them as type 1 regulatory T (Tr1) cells expressing IL-10, granzyme B, perforin, CCL5 and LILRB4. Tumour-specific Tr1 cells suppressed tumour rejection induced by anti-PD1, LDVax or adoptively transferred tumour-specific effector T cells. Mechanistically, HDVax-induced Tr1 cells selectively killed MHC-II tumour antigen-presenting type 1 conventional dendritic cells (cDC1s), leading to low numbers of cDC1s in tumours. We then documented modalities to overcome this inhibition, specifically via anti-LILRB4 blockade, using a CD8-directed IL-2 mutein, or targeted loss of cDC2/monocytes. Collectively, these data show that cytotoxic Tr1 cells, which maintain peripheral tolerance, also inhibit antitumour responses and thereby function to impede immune control of cancer.
Asunto(s)
Antígenos de Neoplasias , Linfocitos T CD4-Positivos , Citotoxicidad Inmunológica , Inmunoterapia , Neoplasias , Linfocitos T Reguladores , Animales , Femenino , Humanos , Masculino , Ratones , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular Tumoral , Quimiocina CCL5/metabolismo , Células Dendríticas/inmunología , Granzimas/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Interleucina-10/metabolismo , Interleucina-10/inmunología , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/terapia , Linfocitos T Reguladores/inmunología , Receptores Inmunológicos/antagonistas & inhibidores , Glicoproteínas de Membrana/antagonistas & inhibidores , Tolerancia Inmunológica , Linfocitos T CD8-positivos/inmunologíaRESUMEN
The ability of the immune system to eliminate and shape the immunogenicity of tumours defines the process of cancer immunoediting1. Immunotherapies such as those that target immune checkpoint molecules can be used to augment immune-mediated elimination of tumours and have resulted in durable responses in patients with cancer that did not respond to previous treatments. However, only a subset of patients benefit from immunotherapy and more knowledge about what is required for successful treatment is needed2-4. Although the role of tumour neoantigen-specific CD8+ T cells in tumour rejection is well established5-9, the roles of other subsets of T cells have received less attention. Here we show that spontaneous and immunotherapy-induced anti-tumour responses require the activity of both tumour-antigen-specific CD8+ and CD4+ T cells, even in tumours that do not express major histocompatibility complex (MHC) class II molecules. In addition, the expression of MHC class II-restricted antigens by tumour cells is required at the site of successful rejection, indicating that activation of CD4+ T cells must also occur in the tumour microenvironment. These findings suggest that MHC class II-restricted neoantigens have a key function in the anti-tumour response that is nonoverlapping with that of MHC class I-restricted neoantigens and therefore needs to be considered when identifying patients who will most benefit from immunotherapy.
Asunto(s)
Antígenos de Neoplasias/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Neoplasias Experimentales/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Inmunoterapia , Ratones , Neoplasias Experimentales/terapiaRESUMEN
Immunotherapies are a promising advance in cancer treatment. However, because only a subset of cancer patients benefits from these treatments it is important to find mechanisms that will broaden the responding patient population. Generally, tumors with high mutational burdens have the potential to express greater numbers of mutant neoantigens. As neoantigens can be targets of protective adaptive immunity, highly mutated tumors are more responsive to immunotherapy. Given that external beam radiation 1) is a standard-of-care cancer therapy, 2) induces expression of mutant proteins and potentially mutant neoantigens in treated cells, and 3) has been shown to synergize clinically with immune checkpoint therapy (ICT), we hypothesized that at least one mechanism of this synergy was the generation of de novo mutant neoantigen targets in irradiated cells. Herein, we use KrasG12D x p53-/- sarcoma cell lines (KP sarcomas) that we and others have shown to be nearly devoid of mutations, are poorly antigenic, are not controlled by ICT, and do not induce a protective antitumor memory response. However, following one in vitro dose of 4- or 9-Gy irradiation, KP sarcoma cells acquire mutational neoantigens and become sensitive to ICT in vivo in a T cell-dependent manner. We further demonstrate that some of the radiation-induced mutations generate cytotoxic CD8+ T cell responses, are protective in a vaccine model, and are sufficient to make the parental KP sarcoma line susceptible to ICT. These results provide a proof of concept that induction of new antigenic targets in irradiated tumor cells represents an additional mechanism explaining the clinical findings of the synergy between radiation and immunotherapy.
Asunto(s)
Antígenos de Neoplasias/inmunología , Inmunoterapia , Mutación/genética , Neoplasias/genética , Neoplasias/inmunología , Radiación , Animales , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Células Clonales , Femenino , Antígenos de Histocompatibilidad Clase II/metabolismo , Proteínas de Punto de Control Inmunitario/metabolismo , Inmunidad , Ratones , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteína p53 Supresora de Tumor/genética , VacunaciónRESUMEN
BACKGROUND: Floxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. However, an efficient and reliable strategy to flox genomic regions of any desired size is still lacking. RESULTS: Here, we demonstrate that the method combining electroporation of fertilized eggs with gRNA/Cas9 complexes and single-stranded oligodeoxynucleotides (ssODNs), assessing phasing of loxP insertions in founders using an in vitro Cre assay and an optional, highly specific and efficient second-round targeting ensures the generation of floxed F1 animals in roughly five months for a wide range of sequence lengths (448 bp to 160 kb reported here). CONCLUSIONS: Floxed alleles can be reliably obtained in a predictable timeline using the improved method of electroporation of two gRNA/Cas9 ribonucleoprotein particles (RNPs) and two ssODNs.
Asunto(s)
Sistemas CRISPR-Cas , ARN Guía de Kinetoplastida , Alelos , Animales , Ratones , Ratones Noqueados , ARN Guía de Kinetoplastida/genética , CigotoRESUMEN
Intricate processes in the thymus and periphery help curb the development and activation of autoreactive T cells. The subtle signals that govern these processes are an area of great interest, but tuning TCR sensitivity for the purpose of affecting T cell behavior remains technically challenging. Previously, our laboratory described the derivation of two TCR-transgenic CD4 T cell mouse lines, LLO56 and LLO118, which recognize the same cognate Listeria epitope with the same affinity. Despite the similarity of the two TCRs, LLO56 cells respond poorly in a primary infection whereas LLO118 cells respond robustly. Phenotypic examination of both lines revealed a substantial difference in their surface of expression of CD5, which serves as a dependable readout of the self-reactivity of a cell. We hypothesized that the increased interaction with self by the CD5-high LLO56 was mediated through TCR signaling, and was involved in the characteristic weak primary response of LLO56 to infection. To explore this issue, we generated an inducible knock-in mouse expressing the self-sensitizing voltage-gated sodium channel Scn5a. Overexpression of Scn5a in peripheral T cells via the CD4-Cre promoter resulted in increased TCR-proximal signaling. Further, Scn5a-expressing LLO118 cells, after transfer into BL6 recipient mice, displayed an impaired response during infection relative to wild-type LLO118 cells. In this way, we were able to demonstrate that tuning of TCR sensitivity to self can be used to alter in vivo immune responses. Overall, these studies highlight the critical relationship between TCR-self-pMHC interaction and an immune response to infection.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Animales , Antígenos CD5/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Canal de Sodio Activado por Voltaje NAV1.5/inmunología , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Natural killer (NK) cells express MHC class I (MHC-I)-specific receptors, such as Ly49A, that inhibit killing of cells expressing self-MHC-I. Self-MHC-I also "licenses" NK cells to become responsive to activating stimuli and regulates the surface level of NK-cell inhibitory receptors. However, the mechanisms of action resulting from these interactions of the Ly49s with their MHC-I ligands, particularly in vivo, have been controversial. Definitive studies could be derived from mice with targeted mutations in inhibitory Ly49s, but there are inherent challenges in specifically altering a single gene within a multigene family. Herein, we generated a knock-in mouse with a targeted mutation in the immunoreceptor tyrosine-based inhibitory motif (ITIM) of Ly49A that abolished the inhibitory function of Ly49A in cytotoxicity assays. This mutant Ly49A caused a licensing defect in NK cells, but the surface expression of Ly49A was unaltered. Moreover, NK cells that expressed this mutant Ly49A exhibited an altered inhibitory receptor repertoire. These results demonstrate that Ly49A ITIM signaling is critical for NK-cell effector inhibition, licensing, and receptor repertoire development.
Asunto(s)
Citotoxicidad Inmunológica/inmunología , Genes MHC Clase I/inmunología , Motivo de Inhibición del Inmunorreceptor Basado en Tirosina , Células Asesinas Naturales/inmunología , Subfamilia A de Receptores Similares a Lectina de Células NK/fisiología , Receptores Similares a Lectina de Células NK/metabolismo , Animales , Células Cultivadas , Células Asesinas Naturales/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptores Similares a Lectina de Células NK/genética , Tirosina/metabolismoRESUMEN
Cancer immunoediting, the process by which the immune system controls tumour outgrowth and shapes tumour immunogenicity, is comprised of three phases: elimination, equilibrium and escape. Although many immune components that participate in this process are known, its underlying mechanisms remain poorly defined. A central tenet of cancer immunoediting is that T-cell recognition of tumour antigens drives the immunological destruction or sculpting of a developing cancer. However, our current understanding of tumour antigens comes largely from analyses of cancers that develop in immunocompetent hosts and thus may have already been edited. Little is known about the antigens expressed in nascent tumour cells, whether they are sufficient to induce protective antitumour immune responses or whether their expression is modulated by the immune system. Here, using massively parallel sequencing, we characterize expressed mutations in highly immunogenic methylcholanthrene-induced sarcomas derived from immunodeficient Rag2(-/-) mice that phenotypically resemble nascent primary tumour cells. Using class I prediction algorithms, we identify mutant spectrin-ß2 as a potential rejection antigen of the d42m1 sarcoma and validate this prediction by conventional antigen expression cloning and detection. We also demonstrate that cancer immunoediting of d42m1 occurs via a T-cell-dependent immunoselection process that promotes outgrowth of pre-existing tumour cell clones lacking highly antigenic mutant spectrin-ß2 and other potential strong antigens. These results demonstrate that the strong immunogenicity of an unedited tumour can be ascribed to expression of highly antigenic mutant proteins and show that outgrowth of tumour cells that lack these strong antigens via a T-cell-dependent immunoselection process represents one mechanism of cancer immunoediting.
Asunto(s)
Exoma/genética , Exoma/inmunología , Vigilancia Inmunológica/inmunología , Neoplasias/genética , Neoplasias/inmunología , Linfocitos T/inmunología , Algoritmos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Masculino , Metilcolantreno , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/inmunología , Modelos Inmunológicos , Neoplasias/inducido químicamente , Neoplasias/patología , Reproducibilidad de los Resultados , Sarcoma/inducido químicamente , Sarcoma/genética , Sarcoma/inmunología , Sarcoma/patologíaAsunto(s)
Formación de Anticuerpos/inmunología , Carcinogénesis/inmunología , Interferón gamma/inmunología , Linfocitos/inmunología , Neoplasias/inmunología , Neoplasias/patología , Animales , Carcinogénesis/patología , Humanos , Huésped Inmunocomprometido/inmunología , Vigilancia Inmunológica/inmunología , Linfocitos/patologíaRESUMEN
Although IFN-γ is required for resolution of Listeria monocytogenes infection, the identities of the IFN-γ-responsive cells that initiate the process remain unclear. We addressed this question using novel mice with conditional loss of IFN-γR (IFNGR1). Itgax-cre(+)Ifngr1(f/f) mice with selective IFN-γ unresponsiveness in CD8α(+) dendritic cells displayed increased susceptibility to infection. This phenotype was due to the inability of IFN-γ-unresponsive CD8α(+) dendritic cells to produce the initial burst of IL-12 induced by IFN-γ from TNF-α-activated NK/NKT cells. The defect in early IL-12 production resulted in increased IL-4 production that established a myeloid cell environment favoring Listeria growth. Neutralization of IL-4 restored Listeria resistance in Itgax-cre(+)Ifngr1(f/f) mice. We also found that Itgax-cre(+)Ifngr1(f/f) mice survived infection with low-dose Listeria as the result of a second wave of IL-12 produced by Ly6C(hi) monocytes. Thus, an IFN-γ-driven cascade involving CD8α(+) dendritic cells and NK/NKT cells induces the rapid production of IL-12 that initiates the anti-Listeria response.
Asunto(s)
Interferón gamma/inmunología , Interleucina-12/inmunología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Receptores de Interferón/inmunología , Animales , Antígenos Ly/metabolismo , Antígenos CD8/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Interleucina-12/biosíntesis , Interleucina-4/biosíntesis , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Factor de Necrosis Tumoral alfa/metabolismo , Receptor de Interferón gammaRESUMEN
The contribution of placental immune responses to congenital Zika virus (ZIKV) syndrome remains poorly understood. Here, we leveraged a mouse model of ZIKV infection to identify mechanisms of innate immune restriction exclusively in the fetal compartment of the placenta. ZIKV principally infected mononuclear trophoblasts in the junctional zone, which was limited by mitochondrial antiviral-signaling protein (MAVS) and type I interferon (IFN) signaling mechanisms. Single nuclear RNA sequencing revealed MAVS-dependent expression of IFN-stimulated genes (ISGs) in spongiotrophoblasts but not in other placental cells that use alternate pathways to induce ISGs. ZIKV infection of Ifnar1-/- or Mavs-/- placentas was associated with greater infection of the adjacent immunocompetent decidua, and heterozygous Mavs+/- or Ifnar1+/- dams carrying immunodeficient fetuses sustained greater maternal viremia and tissue infection than dams carrying wild-type fetuses. Thus, MAVS-IFN signaling in the fetus restricts ZIKV infection in junctional zone trophoblasts, which modulates dissemination and outcome for both the fetus and the pregnant mother.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Decidua , Feto , Interferón Tipo I , Placenta , Receptor de Interferón alfa y beta , Transducción de Señal , Trofoblastos , Infección por el Virus Zika , Virus Zika , Femenino , Animales , Embarazo , Interferón Tipo I/metabolismo , Interferón Tipo I/inmunología , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Placenta/inmunología , Placenta/virología , Placenta/metabolismo , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virología , Virus Zika/inmunología , Virus Zika/fisiología , Ratones , Decidua/inmunología , Decidua/virología , Decidua/metabolismo , Feto/inmunología , Feto/virología , Trofoblastos/inmunología , Trofoblastos/virología , Trofoblastos/metabolismo , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Inmunidad Innata , Complicaciones Infecciosas del Embarazo/inmunología , Complicaciones Infecciosas del Embarazo/virología , Modelos Animales de EnfermedadRESUMEN
Floxed alleles and Cre drivers are two components of most conditional knockout mouse models, which are not only important for studying a given gene in a tissue-specific manner, but also useful for functional analysis of various sized genomic regions. With the increased demand for floxed mouse models in biomedical research, reliable and economical creation of floxed alleles is clearly highly valuable yet remains challenging. Here we provide technical details on the method consisting of electroporating single-cell embryos with CRISPR RNPs and ssODNs, next-generation sequencing (NGS)-based genotyping, an in vitro Cre assay (recombination followed by PCR) for loxP phasing determination, and optional second round targeting of an indel in cis with one loxP insertion in embryos obtained via in vitro fertilization (IVF). As importantly, we present protocols for validation of gRNAs and ssODNs before electroporation of embryos, to confirm phasing of loxP and the indel to be retargeted in individual blastocysts and an alternative strategy to insert loxP sites sequentially. Together, we hope to help researchers reliably obtain floxed alleles in a predictable and timely manner.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Electroporación , Ratones , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Ratones Noqueados , Cigoto , Alelos , Integrasas/genéticaRESUMEN
Rift Valley fever virus (RVFV) is an emerging arbovirus found in Africa. While RVFV is pantropic and infects many cells and tissues, viral replication and necrosis within the liver play a critical role in mediating severe disease. The low-density lipoprotein receptor-related protein 1 (Lrp1) is a recently identified host factor for cellular entry and infection by RVFV. The biological significance of Lrp1, including its role in hepatic disease in vivo, however, remains to be determined. Because Lrp1 has a high expression level in hepatocytes, we developed a mouse model in which Lrp1 is specifically deleted in hepatocytes to test how the absence of liver Lrp1 expression affects RVF pathogenesis. Mice lacking Lrp1 expression in hepatocytes showed minimal RVFV replication in the liver, longer time to death, and altered clinical signs toward neurological disease. In contrast, RVFV infection levels in other tissues showed no difference between the two genotypes. Therefore, Lrp1 is essential for RVF hepatic disease in mice.
Asunto(s)
Fiebre del Valle del Rift , Virus de la Fiebre del Valle del Rift , Animales , Ratones , Fiebre del Valle del Rift/genética , Virus de la Fiebre del Valle del Rift/genética , África , Hepatocitos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genéticaRESUMEN
Twenty-seven subjects with squamous cell cancer of the head and neck received the neoadjuvant IRX-2 immunotherapy regimen prior to surgery in a Phase 2 trial. Pretreatment tumor biopsies were compared with the primary tumor surgical specimens for lymphocyte infiltration, necrosis and fibrosis, using hematoxylin and eosin stain and immunohistochemistry in 25 subjects. Sections were examined by three pathologists. Relative to pretreatment biopsies, increases in lymphocyte infiltration (LI) were seen using H and E or immunohistochemistry. CD3+ CD4+ T cells and CD20+ B cells were primarily found in the peritumoral stroma and CD3+ CD8+ T cells and CD68+ macrophages were mainly intratumoral. LI in the surgical specimens were associated with reductions in the primary tumor size. Improved survival at 5 years was correlated with high overall LI in the tumor specimens. Neoadjuvant IRX-2 immunotherapy regimen may restore immune responsiveness presumably by mobilizing tumor infiltrating effector lymphocytes and macrophages into the tumor.
Asunto(s)
Citocinas/inmunología , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/terapia , Inmunoterapia , Linfocitos Infiltrantes de Tumor/inmunología , Adulto , Anciano , Citocinas/administración & dosificación , Femenino , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Persona de Mediana Edad , Análisis de Regresión , Análisis de SupervivenciaRESUMEN
Fetal and adult hematopoietic stem cells (HSCs) have distinct proliferation rates, lineage biases, gene expression profiles, and gene dependencies. Although these differences are widely recognized, it is not clear how the transition from fetal to adult identity is coordinated. Here we show that murine HSCs and committed hematopoietic progenitor cells (HPCs) undergo a gradual, rather than precipitous, transition from fetal to adult transcriptional states. The transition begins prior to birth and is punctuated by a late prenatal spike in type I interferon signaling that promotes perinatal HPC expansion and sensitizes progenitors to the leukemogenic FLT3ITD mutation. Most other changes in gene expression and enhancer activation are imprecisely timed and poorly coordinated. Thus, heterochronic enhancer elements, and their associated transcripts, are activated independently of one another rather than as part of a robust network. This simplifies the regulatory programs that guide neonatal HSC/HPC ontogeny, but it creates heterogeneity within these populations.
Asunto(s)
Células Madre Adultas , Análisis de la Célula Individual , Animales , Hematopoyesis , Células Madre Hematopoyéticas , Ratones , Transducción de Señal , TranscriptomaRESUMEN
The PA200 proteasome activator is a broadly expressed nuclear protein. Although how PA200 normally functions is not fully understood, it has been suggested to be involved in the repair of DNA double-strand breaks (DSBs). The PA200 gene (Psme4) is composed of 45 coding exons spanning 108 kb on mouse chromosome 11. We generated a PA200 null allele (PA200(Delta)) through Cre-loxP-mediated interchromosomal recombination after targeting loxP sites at either end of the locus. PA200(Delta/Delta) mice are viable and have no obvious developmental abnormalities. Both lymphocyte development and immunoglobulin class switching, which rely on the generation and repair of DNA DSBs, are unperturbed in PA200(Delta/Delta) mice. Additionally, PA200(Delta/Delta) embryonic stem cells do not exhibit increased sensitivity to either ionizing radiation or bleomycin. Thus, PA200 is not essential for the repair of DNA DSBs generated in these settings. Notably, loss of PA200 led to a marked reduction in male, but not female, fertility. This was due to defects in spermatogenesis observed in meiotic spermatocytes and during the maturation of postmeiotic haploid spermatids. Thus, PA200 serves an important nonredundant function during spermatogenesis, suggesting that the efficient generation of male gametes has distinct protein metabolic requirements.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/fisiología , Espermatogénesis , Adenoviridae/genética , Alelos , Animales , Apoptosis , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Cromosomas de los Mamíferos , Exones , Citometría de Flujo , Marcación de Gen , Infertilidad Masculina/genética , Masculino , Meiosis , Ratones , Ratones Noqueados , Modelos Biológicos , Complejo de la Endopetidasa Proteasomal/genética , Recombinación Genética , Espermatocitos/citología , Células Madre/citología , Testículo/citologíaRESUMEN
Herein we report the generation of mouse monoclonal antibodies (mAbs) specific for the IFNAR-1 subunit of the mouse interferon-alpha/beta (IFN-alpha/beta) receptor (MAR1 mAbs) that block type I IFN receptor signaling and biologic response induction in vitro and in vivo. These mAbs were generated from Ifnar1 (/) mice immunized by in vivo hydrodynamic transfection with a plasmid encoding the extracellular domain (ECD) of murine IFNAR-1. All MAR1 mAbs bound native receptor expressed on cell surfaces and immunoprecipitated IFNAR-1 from solubilized cells, and two mAbs also detected IFNAR-1 by Western blot analysis. in vitro, the mAbs prevented ligand-induced intracellular signaling and induction of a variety of type I IFN-induced biologic responses but had no effect on IFN-gamma-induced responses. The most effective in vitro blocker, MAR1-5A3, also blocked type I IFN-induced antiviral, antimicrobial, and antitumor responses in vivo. We also explored whether murine IFNAR-1 surface expression required the presence of Tyk2. In contrast to Tyk2-deficient human cell lines, comparable IFNAR-1 expression was found on primary cells derived either from wild-type or Tyk2 (/) mice. These mAbs represent much needed tools to more clearly elucidate the biochemistry, cell biology, and physiologic function of the type I IFNs and their receptor in mediating host-protective immunity and immunopathology.
Asunto(s)
Anticuerpos Bloqueadores/inmunología , Anticuerpos Monoclonales/inmunología , Receptor de Interferón alfa y beta/inmunología , Animales , Anticuerpos Bloqueadores/aislamiento & purificación , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos , Expresión Génica , Inmunización/métodos , Ratones , Ratones Noqueados , Plásmidos/genética , Plásmidos/inmunología , Receptor de Interferón alfa y beta/antagonistas & inhibidores , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/genética , TYK2 Quinasa/deficiencia , TYK2 Quinasa/inmunología , Transfección/métodosRESUMEN
Focal segmental glomerulosclerosis (FSGS) is a syndrome that involves kidney podocyte dysfunction and causes chronic kidney disease. Multiple factors including chemical toxicity, inflammation, and infection underlie FSGS; however, highly penetrant disease genes have been identified in a small fraction of patients with a family history of FSGS. Variants of apolipoprotein L1 (APOL1) have been linked to FSGS in African Americans with HIV or hypertension, supporting the proposal that genetic factors enhance FSGS susceptibility. Here, we used sequencing to investigate whether genetics plays a role in the majority of FSGS cases that are identified as primary or sporadic FSGS and have no known cause. Given the limited number of biopsy-proven cases with ethnically matched controls, we devised an analytic strategy to identify and rank potential candidate genes and used an animal model for validation. Nine candidate FSGS susceptibility genes were identified in our patient cohort, and three were validated using a high-throughput mouse method that we developed. Specifically, we introduced a podocyte-specific, doxycycline-inducible transactivator into a murine embryonic stem cell line with an FSGS-susceptible genetic background that allows shRNA-mediated targeting of candidate genes in the adult kidney. Our analysis supports a broader role for genetic susceptibility of both sporadic and familial cases of FSGS and provides a tool to rapidly evaluate candidate FSGS-associated genes.
Asunto(s)
Glomeruloesclerosis Focal y Segmentaria/genética , Animales , Estudios de Casos y Controles , Células Cultivadas , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Ratones Transgénicos , Polimorfismo de Nucleótido SimpleRESUMEN
Over the last 12 yr, we have shown that interferon-gamma and lymphocytes collaborate to regulate tumor development in mice. Specifically, we found that the immune system not only prevents the growth of primary (carcinogen-induced and spontaneous) and transplanted tumors but also sculpts the immunogenicity of tumors that form. These observations led us to refine the old and controversial "cancer immunosurveillance" hypothesis of Burnet and Thomas into one that we termed cancer immunoediting that better emphasizes the paradoxical host-protective and tumor-sculpting roles of immunity on developing tumors. Our current work focuses on defining the molecular mechanisms that underlie cancer immunoediting and exploring the implications of this process for cancer immunotherapy.
Asunto(s)
Interferón gamma/inmunología , Neoplasias/inmunología , Animales , Genes p53 , Rechazo de Injerto/inmunología , Humanos , Inmunoterapia , Linfocitos/inmunología , Ratones , Ratones Noqueados , Modelos Inmunológicos , Monitorización Inmunológica , Trasplante de Neoplasias , Neoplasias/prevención & control , Neoplasias/terapia , Transducción de Señal/inmunología , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
The bacterial CRISPR-Cas9 system has been adapted for use as a genome editing tool. While several recent reports have indicated that successful genome editing of mice can be achieved, detailed phenotypic and molecular analyses of the mutant animals are limited. Following pronuclear micro-injection of fertilized eggs with either wild-type Cas9 or the nickase mutant (D10A) and single or paired guide RNA (sgRNA) for targeting of the tyrosinase (Tyr) gene, we assessed genome editing in mice using rapid phenotypic readouts (eye and coat color). Mutant mice with insertions or deletions (indels) in Tyr were efficiently generated without detectable off-target cleavage events. Gene correction of a single nucleotide by homologous recombination (HR) could only occur when the sgRNA recognition sites in the donor DNA were modified. Gene repair did not occur if the donor DNA was not modified because Cas9 catalytic activity was completely inhibited. Our results indicate that allelic mosaicism can occur following -Cas9-mediated editing in mice and appears to correlate with sgRNA cleavage efficiency at the single-cell stage. We also show that larger than expected deletions may be overlooked based on the screening strategy employed. An unbiased analysis of all the deleted nucleotides in our experiments revealed that the highest frequencies of nucleotide deletions were clustered around the predicted Cas9 cleavage sites, with slightly broader distributions than expected. Finally, additional analysis of founder mice and their offspring indicate that their general health, fertility, and the transmission of genetic changes were not compromised. These results provide the foundation to interpret and predict the diverse outcomes following CRISPR-Cas9-mediated genome editing experiments in mice.
Asunto(s)
Sistemas CRISPR-Cas , Monofenol Monooxigenasa/genética , Fenotipo , Alelos , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Genoma , Ratones , Ratones Transgénicos , ARN Guía de KinetoplastidaRESUMEN
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide and results from a complex interaction between carcinogen exposure and inherent susceptibility. Despite its prevalence, genetic factors that predispose to the development of lung cancer remain elusive. Inbred mouse models offer a unique and clinically relevant tool to study genetic factors that contribute to lung carcinogenesis due to the development of tumors that resemble human adenocarcinoma and broad strain-specific variation in cancer incidence after carcinogen administration. Here, we set out to investigate whether strain-specific variability in tumor immunosurveillance contributes to differences in lung cancer. Using bone marrow transplantation, we determined that hematopoietic cells from lung cancer-resistant mice could significantly impede the development of cancer in a susceptible strain. Furthermore, we show that this is not due to differences in tumor-promoting inflammatory changes or variability in immunosurveillance by the adaptive immune system but results from strain-specific differences in natural killer (NK) cell cytotoxicity. Using a newly discovered congenic strain of mice, we show a previously unrecognized role for strain-specific polymorphisms in the natural killer gene complex (NKC) in immunosurveillance for carcinogen-induced lung cancer. Because polymorphisms in the NKC are highly prevalent in man, our data may explain why certain individuals without obvious risk factors develop lung cancer whereas others remain resistant to the disease despite heavy environmental carcinogen exposure.