RESUMEN
Stem cells are maintained by transcriptional programs that promote self-renewal and repress differentiation. Here, we found that the transcription factor c-Myb was essential for generating and maintaining stem cells in the CD8+ T cell memory compartment. Following viral infection, CD8+ T cells lacking Myb underwent terminal differentiation and generated fewer stem cell-like central memory cells than did Myb-sufficient T cells. c-Myb acted both as a transcriptional activator of Tcf7 (which encodes the transcription factor Tcf1) to enhance memory development and as a repressor of Zeb2 (which encodes the transcription factor Zeb2) to hinder effector differentiation. Domain-mutagenesis experiments revealed that the transactivation domain of c-Myb was necessary for restraining differentiation, whereas its negative regulatory domain was critical for cell survival. Myb overexpression enhanced CD8+ T cell memory formation, polyfunctionality and recall responses that promoted curative antitumor immunity after adoptive transfer. These findings identify c-Myb as a pivotal regulator of CD8+ T cell stemness and highlight its therapeutic potential.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Neoplasias Experimentales/inmunología , Proteínas Proto-Oncogénicas c-myb/inmunología , Células Madre/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Células HEK293 , Humanos , Memoria Inmunológica/genética , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/virología , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Células Madre/metabolismo , Células Madre/virología , Factor 1 de Transcripción de Linfocitos T/genética , Factor 1 de Transcripción de Linfocitos T/inmunología , Factor 1 de Transcripción de Linfocitos T/metabolismoRESUMEN
Maintaining antitumor immunity remains a persistent impediment to cancer immunotherapy. We and others have previously reported that high-avidity CD8(+) T cells are more susceptible to tolerance induction in the tumor microenvironment. In the present study, we used a novel model where T cells derived from two independent TCR transgenic mouse lines recognize the same melanoma antigenic epitope but differ in their avidity. We tested whether providing CD4(+) T cell help would improve T cell responsiveness as a function of effector T cell avidity. Interestingly, delivery of CD4(+) T cell help during in vitro priming of CD8(+) T cells improved cytokine secretion and lytic capacity of high-avidity T cells, but not low-avidity T cells. Consistent with this observation, copriming with CD4(+) T cells improved antitumor immunity mediated by higher avidity, melanoma-specific CD8(+) T cells, but not T cells with similar specificity but lower avidity. Enhanced tumor immunity was associated with improved CD8(+) T cell expansion and reduced tolerization, and it was dependent on presentation of both CD4(+) and CD8(+) T cell epitopes by the same dendritic cell population. Our findings demonstrate that CD4(+) T cell help preferentially augments high-avidity CD8(+) T cells and provide important insight for understanding the requirements to elicit and maintain durable tumor immunity.
Asunto(s)
Afinidad de Anticuerpos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica/inmunología , Tolerancia Inmunológica/inmunología , Animales , Afinidad de Anticuerpos/genética , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Células Dendríticas/inmunología , Epítopos de Linfocito T/inmunología , Activación de Linfocitos/inmunología , Melanoma/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the intestine that result in painful and debilitating complications. Currently no cure exists for IBD, and treatments are primarily aimed at reducing inflammation to alleviate symptoms. Genome-wide linkage studies have identified the Ron receptor tyrosine kinase (TK) and its ligand, hepatocyte growth factor-like protein (HGFL), as genes highly associated with IBD. However, only scant information exists on the role of Ron or HGFL in IBD. Based on the linkage of Ron to IBD, we directly examined the biological role of Ron in colitis. Wild-type mice and mice lacking the TK signaling domain of Ron (TK-/- mice) were utilized in a well-characterized model of chronic colitis induced by cyclic exposure to dextran sulfate sodium. In this model, TK-/- mice were more susceptible to injury as judged by increased mortality compared with control mice and developed more severe colitis. Loss of Ron led to significantly reduced body weights and more aggressive clinical and histopathologies. Ron loss also resulted in a dramatic reduction in colonic epithelial cell proliferation and increased proinflammatory cytokine production, which was associated with alterations in important signaling pathways known to regulate IBD. Examination of human gene expression data further supports the contention that loss of Ron signaling is associated with IBD. In total, our studies point to important functional roles for Ron in IBD by regulating healing of the colonic epithelium and by controlling cytokine secretion.
Asunto(s)
Colitis/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/fisiología , Animales , Colitis/inducido químicamente , Sulfato de Dextran , Modelos Animales de Enfermedad , Expresión Génica/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
BACKGROUND: Metastatic cholangiocarcinoma (CC), a form of gastrointestinal cancer that originates from the bile ducts, cannot be cured by currently available therapies, and is associated with dismal prognosis. In a previous case report, adoptive transfer of autologous tumor infiltrating lymphocytes (TILs), the majority of which recognized a tumor-specific point mutation, led to a profound and durable cancer regression in a patient with metastatic CC. Thus, more effective treatment for patients with this disease may be developed by using TILs that target cancer-specific mutations, but also other genetic aberrations such as gene fusions. In this context, fusions that involve fibroblast growth factor receptor 2 (FGFR2) and function as oncogenes in a subset of patients with intrahepatic CC (ICC) represent particularly attractive targets for adoptive cell therapy. However, no study to date has explored whether FGFR2 fusions can be recognized by patients' T cells. METHOD: To address whether FGFR2 fusions can be recognized by patients' T cells, we tested TILs from four patients with FGFR2 fusion-positive ICC for recognition of peptides and minigenes that represented the breakpoint regions of these fusions, which were unique to each of the four patients. RESULTS: We found that CD4+ TILs from one patient specifically recognized the breakpoint region of a unique FGFR2-TDRD1 (tudor domain-containing 1) fusion, and we isolated a T-cell receptor responsible for its recognition. CONCLUSIONS: This finding suggests that FGFR2 fusion-reactive TILs can be isolated from some patients with metastatic ICC, and thus provides a rationale for future exploration of T cell-based therapy targeting FGFR2 fusions in patients with cancer. Furthermore, it augments the rationale for extending such efforts to other types of solid tumors hallmarked by oncogenic gene fusions.
Asunto(s)
Neoplasias de los Conductos Biliares , Colangiocarcinoma , Humanos , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Conductos Biliares Intrahepáticos/metabolismo , Conductos Biliares Intrahepáticos/patología , Colangiocarcinoma/patología , Linfocitos Infiltrantes de Tumor/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismoRESUMEN
BACKGROUND: The receptor tyrosine kinase family includes many transmembrane proteins with diverse physiological and pathophysiological functions. The involvement of tyrosine kinase signaling in promoting a more aggressive tumor phenotype within the context of chemotherapeutic evasion is gaining recognition. The Ron receptor is a tyrosine kinase receptor that has been implicated in the progression of breast cancer and evasion of tamoxifen therapy. RESULTS: Here, we report that Ron expression is correlated with in situ, estrogen receptor alpha (ERα)-positive tumors, and is higher in breast tumors following neoadjuvant tamoxifen therapy. We also demonstrate that the majority of mammary tumors isolated from transgenic mice with mammary specific-Ron overexpression (MMTV-Ron mice), exhibit appreciable ER expression. Moreover, genetic-ablation of ERα, in the context of Ron overexpression, leads to delayed mammary tumor initiation and growth, but also results in an increased metastasis. CONCLUSIONS: Ron receptor overexpression is associated with ERα-positive human and murine breast tumors. In addition, loss of ERα on a Ron overexpressing background in mice leads to the development of breast tumors which grow slower but which exhibit more metastasis and suggests that targeting of ERα, as in the case of tamoxifen therapy, may reduce the growth of Ron overexpressing breast cancers but may cause these tumors to be more metastatic.
Asunto(s)
Receptor alfa de Estrógeno/genética , Eliminación de Gen , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Proteínas Tirosina Quinasas Receptoras/genética , Animales , Proliferación Celular , Receptor alfa de Estrógeno/metabolismo , Femenino , Expresión Génica , Neoplasias Mamarias Animales/mortalidad , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Metástasis de la Neoplasia , Estadificación de Neoplasias , Fenotipo , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
Adoptive T cell therapies (ACT) have been curative for a limited number of cancer patients. The sensitization of cancer cells to T cell killing may expand the benefit of these therapies for more patients. To this end, we use a three-step approach to identify cancer genes that disfavor T cell immunity. First, we profile gene transcripts upregulated by cancer under selection pressure from T cell killing. Second, we identify potential tumor gene targets and pathways that disfavor T cell killing using signaling pathway activation libraries and genome-wide loss-of-function CRISPR-Cas9 screens. Finally, we implement pharmacological perturbation screens to validate these targets and identify BIRC2, ITGAV, DNPEP, BCL2, and ERRα as potential ACT-drug combination candidates. Here, we establish that BIRC2 limits antigen presentation and T cell recognition of tumor cells by suppressing IRF1 activity and provide evidence that BIRC2 inhibition in combination with ACT is an effective strategy to increase efficacy.
Asunto(s)
Neoplasias , Linfocitos T , Presentación de Antígeno , Sistemas CRISPR-Cas/genética , Humanos , Neoplasias/genética , Oncogenes , Análisis de SistemasRESUMEN
BACKGROUND: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade. METHODS: Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade. FINDINGS: CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo. CONCLUSIONS: CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy. FUNDING: This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.
Asunto(s)
Linfocitos Infiltrantes de Tumor , Linfocitos T , Traslado Adoptivo , Animales , Citocinas/metabolismo , Humanos , Inmunoterapia Adoptiva/métodos , RatonesRESUMEN
Inflammatory response plays an essential role in the resolution of infections. However, inflammation can be detrimental to an organism and cause irreparable damage. For example, during sepsis, a cytokine storm can lead to multiple organ failures and often results in death. One of the strongest triggers of the inflammatory response is bacterial lipopolysaccharides (LPS), acting mostly through Toll-like receptor 4 (TLR4). Paradoxically, while exposure to LPS triggers a robust inflammatory response, repeated or prolonged exposure to LPS can induce a state of endotoxin tolerance, a phenomenon where macrophages and monocytes do not respond to new endotoxin challenges, and it is often associated with secondary infections and negative outcomes. The cellular mechanisms regulating this phenomenon remain elusive. We used metabolic measurements to confirm differences in the cellular metabolism of naïve macrophages and that of macrophages responding to LPS stimulation or those in the LPS-tolerant state. In parallel, we performed an unbiased secretome survey using quantitative mass spectrometry during the induction of LPS tolerance, creating the first comprehensive secretome profile of endotoxin-tolerant cells. The secretome changes confirmed that LPS-tolerant macrophages have significantly decreased cellular metabolism and that the proteins secreted by LPS-tolerant macrophages have a strong association with cell survival, protein metabolism, and the metabolism of reactive oxygen species.
Asunto(s)
Citocinas/metabolismo , Macrófagos/metabolismo , Receptor Toll-Like 4/genética , Animales , Respiración de la Célula/efectos de los fármacos , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Inflamación , Espectrometría de Masas , Ratones , Monocitos/metabolismo , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/metabolismoRESUMEN
The adoptive transfer of naturally occurring T cells that recognize cancer neoantigens has led to durable tumor regressions in select patients with cancer. However, it remains unknown whether such T cells can be isolated from and used to treat patients with glioblastoma, a cancer that is refractory to currently available therapies. To answer this question, we stimulated patient blood-derived memory T cells in vitro using peptides and minigenes that represented point mutations unique to patients' tumors (ie, candidate neoantigens) and then tested their ability to specifically recognize these mutations. In a cohort of five patients with glioblastoma, we found that circulating CD4+ memory T cells from one patient recognized a cancer neoantigen harboring a mutation in the EED gene (EEDH189N) that was unique to that patient's tumor. This finding suggests that neoantigen-reactive T cells could indeed be isolated from patients with glioblastoma, thereby providing a rationale for further efforts to develop neoantigen-directed adoptive T cell therapy for this disease.
Asunto(s)
Glioblastoma/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , HumanosRESUMEN
Adoptive T cell therapy (ACT) using ex vivo-expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate complete regression of certain human cancers. The impact of TIL phenotypes on clinical success of TIL-ACT is currently unclear. Using high-dimensional analysis of human ACT products, we identified a memory-progenitor CD39-negative stem-like phenotype (CD39-CD69-) associated with complete cancer regression and TIL persistence and a terminally differentiated CD39-positive state (CD39+CD69+) associated with poor TIL persistence. Most antitumor neoantigen-reactive TILs were found in the differentiated CD39+ state. However, ACT responders retained a pool of CD39- stem-like neoantigen-specific TILs that was lacking in ACT nonresponders. Tumor-reactive stem-like TILs were capable of self-renewal, expansion, persistence, and superior antitumor response in vivo. These data suggest that TIL subsets mediating ACT response are distinct from TIL subsets enriched for antitumor reactivity.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunoterapia Adoptiva/métodos , Linfocitos Infiltrantes de Tumor/trasplante , Melanoma/terapia , Neoplasias Cutáneas/terapia , Animales , Antígenos CD/análisis , Antígenos de Diferenciación de Linfocitos T/análisis , Apirasa/análisis , Linfocitos T CD8-positivos/química , Femenino , Humanos , Lectinas Tipo C/análisis , Melanoma/inmunología , Ratones , Ratones Mutantes , Neoplasias Cutáneas/inmunologíaRESUMEN
Barrett's esophagus (BE) is associated with reflux and is implicated the development of esophageal adenocarcinoma (EAC). Apoptosis induces cell death through mitochondrial outer membrane permeabilization (MOMP), which is considered an irreversible step in apoptosis. Activation of MOMP to levels that fail to reach the apoptotic threshold may paradoxically promote cancer-a phenomenon called "Minority MOMP." We asked whether reflux-induced esophageal carcinogenesis occurred via minority MOMP and whether compensatory resistance mechanisms prevented cell death during this process. We exposed preneoplastic, hTERT-immortalized Barrett's cell, CP-C and CP-A, to the oncogenic bile acid, deoxycholic acid (DCA), for 1 year. Induction of minority MOMP was tested via comet assay, CyQuant, annexin V, JC-1, cytochrome C subcellular localization, caspase 3 activation, and immunoblots. We used bcl-2 homology domain-3 (BH3) profiling to test the mitochondrial apoptotic threshold. One-year exposure of Barrett's cells to DCA induced a malignant phenotype noted by clone and tumor formation. DCA promoted minority MOMP noted by minimal release of cytochrome C and limited caspase 3 activation, which resulted in DNA damage without apoptosis. Upregulation of the antiapoptotic protein, Mcl-1, ROS generation, and NF-κB activation occurred in conjunction with minority MOMP. Inhibition of ROS blocked minority MOMP and Mcl-1 upregulation. Knockdown of Mcl-1 shifted minority MOMP to complete MOMP as noted by dynamic BH3 profiling and increased apoptosis. Minority MOMP contributes to DCA induced carcinogenesis in preneoplastic BE. Mcl-1 provided a balance within the mitochondria that induced resistance complete MOMP and cell death. Targeting Mcl-1 may be a therapeutic strategy in EAC.
Asunto(s)
Apoptosis , Esófago de Barrett/patología , Ácidos y Sales Biliares/farmacología , Carcinogénesis/patología , Neoplasias Esofágicas/patología , Mitocondrias/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Esófago de Barrett/tratamiento farmacológico , Esófago de Barrett/genética , Esófago de Barrett/metabolismo , Carcinogénesis/inducido químicamente , Carcinogénesis/genética , Carcinogénesis/metabolismo , Línea Celular , Permeabilidad de la Membrana Celular , Citocromos c/metabolismo , Daño del ADN , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Esófago/efectos de los fármacos , Esófago/metabolismo , Esófago/patología , Fármacos Gastrointestinales/farmacología , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Transducción de SeñalRESUMEN
T cells are central to all currently effective cancer immunotherapies, but the characteristics defining therapeutically effective anti-tumor T cells have not been comprehensively elucidated. Here, we delineate four phenotypic qualities of effective anti-tumor T cells: cell expansion, differentiation, oxidative stress, and genomic stress. Using a CRISPR-Cas9-based genetic screen of primary T cells we measured the multi-phenotypic impact of disrupting 25 T cell receptor-driven kinases. We identified p38 kinase as a central regulator of all four phenotypes and uncovered transcriptional and antioxidant pathways regulated by p38 in T cells. Pharmacological inhibition of p38 improved the efficacy of mouse anti-tumor T cells and enhanced the functionalities of human tumor-reactive and gene-engineered T cells, paving the way for clinically relevant interventions.
Asunto(s)
Neoplasias de la Mama/terapia , Sistemas CRISPR-Cas , Inmunoterapia Adoptiva/métodos , Melanoma Experimental/terapia , Fenotipo , Linfocitos T/trasplante , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Diferenciación Celular , Femenino , Ingeniería Genética , Masculino , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
In patients with active lupus, spontaneous endotoxemia and possibly tolerance to lipopolysaccharide (LPS) is a potentially adverse complication. Similarly, previous reports have demonstrated that FcGRIIb deficient mice (FcGRIIb-/-; a lupus mouse model) are susceptible to LPS tolerance-induced decreased cytokine responses that inadequate for the organismal control. Thus, understanding the relationship between FcGRIIb and LPS tolerance could improve the therapeutic strategy for lupus. LPS tolerance can be induced through sequential LPS stimulations in either cells or a model organism. In RAW264.7 (a mouse macrophage cell-line), sequential LPS stimulation induced the secretion of Lipocalin-2 (Lcn-2) despite reduced cytokine secretion and severe energy depletion, as measured by the extracellular flux analysis, typical of LPS tolerance. In contrast, treatment with recombinant Lcn-2 (rLcn-2) attenuated LPS tolerance, as shown by an increase in secreted cytokines and altered macrophage polarization toward M1 (increased iNOS and TNF-α) in RAW264.7 cells. These results suggest a role of Lcn-2 in LPS tolerance attenuation. In bone marrow derived macrophages, Lcn-2 level was similar in LPS tolerant FcGRIIb-/- and wild-type (WT) cells despite the increased LPS tolerance of FcGRIIb-/- cells, suggesting relatively low basal levels of Lcn-2 produced in FcGRIIb-/- cells. In addition, attenuation of LPS tolerance effectuated by granulocyte-monocyte colony stimulating factor (GM-CSF) reduced Lcn-2 in both cell types, implying an inverse correlation between Lcn-2 and the severity of LPS tolerance. Consequently, rLcn-2 improved LPS tolerance only in FcGRIIb-/- macrophages and attenuated disease severity of cecal ligation and puncture (CLP) sepsis pre-conditioning with sequential LPS injection (LPS-CLP model) only in FcGRIIb-/- mice, but not in WT mice. To summarize, inadequate Lcn-2 production in FcGRIIb-/- macrophage might, at least in part, be responsible for the inordinate LPS tolerance compared with WT cells. Additionally, supplementation of rLcn-2 attenuates LPS tolerance in FcGRIIb-/- macrophages in vitro, and in FcGRIIb-/- mice with LPS-CLP sepsis in vivo. In conclusion, Lcn-2 secreted by macrophages is possibly an autocrine signal to counter the reduced cytokine secretion in LPS tolerance.
Asunto(s)
Endotoxemia/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Lipocalina 2 , Lipopolisacáridos/inmunología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Receptores de IgG/inmunología , Animales , Citocinas/inmunología , Modelos Animales de Enfermedad , Endotoxemia/etiología , Lipocalina 2/farmacología , Lipocalina 2/fisiología , Lupus Eritematoso Sistémico/complicaciones , Macrófagos , Ratones , Ratones Endogámicos C57BL , Células RAW 264.7 , Proteínas Recombinantes/farmacologíaRESUMEN
Host conditioning has emerged as an important component of effective adoptive cell transfer-based immunotherapy for cancer. High levels of IL-1ß are induced by host conditioning, but its impact on the antitumor function of T cells remains unclear. We found that the administration of IL-1ß increased the population size and functionality of adoptively transferred T cells within the tumor. Most importantly, IL-1ß enhanced the ability of tumor-specific T cells to trigger the regression of large, established B16 melanoma tumors in mice. Mechanistically, we showed that the increase in T cell numbers was associated with superior tissue homing and survival abilities and was largely mediated by IL-1ß-stimulated host cells. In addition, IL-1ß enhanced T cell functionality indirectly via its actions on radio-resistant host cells in an IL-2- and IL-15-dependent manner. Our findings not only underscore the potential of provoking inflammation to enhance antitumor immunity but also uncover novel host regulations of T cell responses.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Interleucina-1beta/inmunología , Activación de Linfocitos/inmunología , Melanoma Experimental/terapia , Linfocitos T/inmunología , Traslado Adoptivo , Animales , Línea Celular Tumoral , Citocinas/sangre , Interleucina-1beta/metabolismo , Interleucina-6/sangre , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Linfocitos T/metabolismo , Linfocitos T/trasplante , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Across clinical trials, T cell expansion and persistence following adoptive cell transfer (ACT) have correlated with superior patient outcomes. Herein, we undertook a pan-cancer analysis to identify actionable ligand-receptor pairs capable of compromising T cell durability following ACT. We discovered that FASLG, the gene encoding the apoptosis-inducing ligand FasL, is overexpressed within the majority of human tumor microenvironments (TMEs). Further, we uncovered that Fas, the receptor for FasL, is highly expressed on patient-derived T cells used for clinical ACT. We hypothesized that a cognate Fas-FasL interaction within the TME might limit both T cell persistence and antitumor efficacy. We discovered that genetic engineering of Fas variants impaired in the ability to bind FADD functioned as dominant negative receptors (DNRs), preventing FasL-induced apoptosis in Fas-competent T cells. T cells coengineered with a Fas DNR and either a T cell receptor or chimeric antigen receptor exhibited enhanced persistence following ACT, resulting in superior antitumor efficacy against established solid and hematologic cancers. Despite increased longevity, Fas DNR-engineered T cells did not undergo aberrant expansion or mediate autoimmunity. Thus, T cell-intrinsic disruption of Fas signaling through genetic engineering represents a potentially universal strategy to enhance ACT efficacy across a broad range of human malignancies.
Asunto(s)
Traslado Adoptivo , Ingeniería Genética , Neoplasias Experimentales/terapia , Receptores Quiméricos de Antígenos , Transducción de Señal/inmunología , Microambiente Tumoral/inmunología , Animales , Proteína Ligando Fas/genética , Proteína Ligando Fas/inmunología , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/inmunología , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/uso terapéutico , Transducción de Señal/genética , Microambiente Tumoral/genética , Receptor fas/genética , Receptor fas/inmunologíaRESUMEN
Induced pluripotent stem cell (iPSC)-derived T cells may provide future therapies for cancer patients, but those generated by current methods, such as the OP9/DLL1 system, have shown abnormalities that pose major barriers for clinical translation. Our data indicate that these iPSC-derived CD8 single-positive T cells are more like CD4+CD8+ double-positive T cells than mature naive T cells because they display phenotypic markers of developmental arrest and an innate-like phenotype after stimulation. We developed a 3D thymic culture system to avoid these aberrant developmental fates, generating a homogeneous subset of CD8αß+ antigen-specific T cells, designated iPSC-derived thymic emigrants (iTEs). iTEs exhibit phenotypic and functional similarities to naive T cells both in vitro and in vivo, including the capacity for expansion, memory formation, and tumor suppression. These data illustrate the limitations of current methods and provide a tool to develop the next generation of iPSC-based antigen-specific immunotherapies.
Asunto(s)
Imagenología Tridimensional/métodos , Células Madre Pluripotentes Inducidas/citología , Timo/citología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Timo/diagnóstico por imagen , Timo/inmunologíaRESUMEN
Adaptive evolution has prompted immune cells to use a wide variety of inhibitory signals, many of which are usurped by tumor cells to evade immune surveillance. Although tumor immunologists often focus on genes and proteins as mediators of immune function, here we highlight two elements from the periodic table-oxygen and potassium-that suppress the immune system in previously unappreciated ways. While both are key to the maintenance of T-cell function and tissue homeostasis, they are exploited by tumors to suppress immuno-surveillance and promote metastatic spread. We discuss the temporal and spatial roles of these elements within the tumor microenvironment and explore possible therapeutic interventions for effective and promising anticancer therapies. Cancer Immunol Res; 5(6); 426-33. ©2017 AACR.
Asunto(s)
Tolerancia Inmunológica , Microambiente Tumoral/inmunología , Animales , Humanos , Neoplasias/inmunología , Oxígeno/inmunología , Potasio/inmunología , Linfocitos T/inmunologíaRESUMEN
Adoptive immunotherapies using T cells genetically redirected with a chimeric antigen receptor (CAR) or T cell receptor (TCR) are entering mainstream clinical practice. Despite encouraging results, some patients do not respond to current therapies. In part, this phenomenon has been associated with infusion of reduced numbers of early memory T cells. Herein, we report that AKT signaling inhibition is compatible with CAR and TCR retroviral transduction of human T cells while promoting a CD62L-expressing central memory phenotype. Critically, this intervention did not compromise cell yield. Mechanistically, disruption of AKT signaling preserved MAPK activation and promoted the intranuclear localization of FOXO1, a transcriptional regulator of T cell memory. Consequently, AKT signaling inhibition synchronized the transcriptional profile for FOXO1-dependent target genes across multiple donors. Expression of an AKT-resistant FOXO1 mutant phenocopied the influence of AKT signaling inhibition, while addition of AKT signaling inhibition to T cells expressing mutant FOXO1 failed to further augment the frequency of CD62L-expressing cells. Finally, treatment of established B cell acute lymphoblastic leukemia was superior using anti-CD19 CAR-modified T cells transduced and expanded in the presence of an AKT inhibitor compared with conventionally grown T cells. Thus, inhibition of signaling along the PI3K/AKT axis represents a generalizable strategy to generate large numbers of receptor-modified T cells with an early memory phenotype and superior antitumor efficacy.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Proteínas Proto-Oncogénicas c-akt/inmunología , Receptores Quiméricos de Antígenos/inmunología , Subgrupos de Linfocitos T/inmunología , Ingeniería de Tejidos/métodos , Animales , Diferenciación Celular , Femenino , Proteína Forkhead Box O1/metabolismo , Regulación de la Expresión Génica/inmunología , Humanos , Memoria Inmunológica , Selectina L/metabolismo , Activación de Linfocitos/inmunología , Ratones Endogámicos NOD , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/inmunología , Transducción Genética/métodos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Ron receptor tyrosine kinase regulates multiple cellular processes and is important during mammary gland development and tumor progression. Hepatocyte growth factor-like protein [HGFL] is the only known ligand for the Ron receptor and recent studies have identified major roles for HGFL during breast cancer metastasis. Understanding the functional importance HGFL during mammary gland development will provide significant insights onto its contribution during tumor development and metastasis. In this study, we assessed the role of HGFL during postnatal mammary gland development using mice that were either proficient [HGFL +/+] or deficient [HGFL-/-] for HGFL. Postnatal ductal morphology and stromal cell associations were analyzed at multiple time points through puberty until adulthood. HGFL deficiency resulted in several mammary gland developmental defects including smaller terminal end buds [TEBs], significantly fewer TEBs, and delayed ductal outgrowth during early puberty. Additionally, HGFL deficient animals exhibited significantly altered TEB epithelial cell turnover with decreased proliferation and increased apoptosis coupled with decreased TEB diameter. Macrophage recruitment to the TEBs was also significantly decreased in the HGFL-/- mice compared to controls. Moreover, the levels of STAT3 mRNA as well as the phosphorylation status of this protein were lower in the HGFL-/- mammary glands compared to controls. Taken together, our data provide the first evidence for HGFL as a positive regulator of mammary gland ductal morphogenesis by controlling overall epithelial cell turnover, macrophage recruitment, and STAT3 activation in the developing mammary gland. With a function in early mammary gland development, HGFL represents a potential target for the development of novel breast cancer therapies.
Asunto(s)
Factor de Crecimiento de Hepatocito/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Factor de Crecimiento de Hepatocito/deficiencia , Factor de Crecimiento de Hepatocito/genética , Macrófagos/citología , Macrófagos/metabolismo , Glándulas Mamarias Animales/citología , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de SeñalRESUMEN
Ron receptor kinase (MST1R) is important in promoting epithelial tumorigenesis, but the potential contributions of its specific expression in stromal cells have not been examined. Herein, we show that the Ron receptor is expressed in mouse and human stromal cells of the prostate tumor microenvironment. To test the significance of stromal Ron expression, prostate cancer cells were orthotopically implanted into the prostates of either wild-type or Ron tyrosine kinase deficient (TK(-/-); Mst1r(-/-)) hosts. In TK(-/-) hosts, prostate cancer cell growth was significantly reduced as compared with tumor growth in TK(+/+) hosts. Prostate tumors in TK(-/-) hosts exhibited an increase in tumor cell apoptosis, macrophage infiltration and altered cytokine expression. Reciprocal bone marrow transplantation studies and myeloid cell-specific ablation of Ron showed that loss of Ron in myeloid cells is sufficient to inhibit prostate cancer cell growth. Interestingly, depletion of CD8(+) T cells, but not CD4(+) T cells, was able to restore prostate tumor growth in hosts devoid of myeloid-specific Ron expression. These studies show a critical role for the Ron receptor in the tumor microenvironment, whereby Ron loss in tumor-associated macrophages inhibits prostate cancer cell growth, at least in part, by derepressing the activity of CD8(+) T cells.