RESUMEN
High frequencies of stem-like memory T cells in infusion products correlate with superior patient outcomes across multiple T cell therapy trials. Herein, we analyzed a published CRISPR activation screening to identify transcriptional regulators that could be harnessed to augment stem-like behavior in CD8+ T cells. Using IFN-γ production as a proxy for CD8+ T cell terminal differentiation, LMO4 emerged among the top hits inhibiting the development of effectors cells. Consistently, we found that Lmo4 was downregulated upon CD8+ T cell activation but maintained under culture conditions facilitating the formation of stem-like T cells. By employing a synthetic biology approach to ectopically express LMO4 in antitumor CD8+ T cells, we enabled selective expansion and enhanced persistence of transduced cells, while limiting their terminal differentiation and senescence. LMO4 overexpression promoted transcriptional programs regulating stemness, increasing the numbers of stem-like CD8+ memory T cells and enhancing their polyfunctionality and recall capacity. When tested in syngeneic and xenograft tumor models, LMO4 overexpression boosted CD8+ T cell antitumor immunity, resulting in enhanced tumor regression. Rather than directly modulating gene transcription, LMO4 bound to JAK1 and potentiated STAT3 signaling in response to IL-21, inducing the expression of target genes (Tcf7, Socs3, Junb, and Zfp36) crucial for memory responses. CRISPR/Cas9-deletion of Stat3 nullified the enhanced memory signature conferred by LMO4, thereby abrogating the therapeutic benefit of LMO4 overexpression. These results establish LMO4 overexpression as an effective strategy to boost CD8+ T cell stemness, providing a new synthetic biology tool to bolster the efficacy of T cell-based immunotherapies.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Linfocitos T CD8-positivos , Proteínas con Dominio LIM , Factor de Transcripción STAT3 , Transducción de Señal , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/inmunología , Linfocitos T CD8-positivos/inmunología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT3/metabolismo , Ratones , Animales , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Humanos , Transducción de Señal/inmunología , Transducción de Señal/genética , Interleucinas/genética , Interleucinas/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunologíaRESUMEN
The functional fitness of CAR T cells plays a crucial role in determining their clinical efficacy. Several strategies are being explored to increase cellular fitness, but screening these approaches in vivo is expensive and time-consuming, limiting the number of strategies that can be tested at one time. The presence of polyfunctional CAR T cells has emerged as a critical parameter correlating with clinical responses. However, even sophisticated multiplexed secretomic assays often fail to detect differences in cytokine release due to the functional heterogeneity of CAR T cell products. Here, we describe a highly multiplexed single-cell secretomic assay based on the IsoLight platform to rapidly evaluate the impact of new pharmacologic or gene-engineering approaches aiming at improving CAR T cell function. As a key study, we focus on CD19-specific CAR CD8+ T cells modulated by miR-155 overexpression, but the protocol can be applied to characterize other functional immune cell modulation strategies.
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Linfocitos T CD8-positivos , Receptores de Antígenos de Linfocitos T , Resultado del Tratamiento , Antígenos CD19 , Inmunoterapia Adoptiva/métodosRESUMEN
Interleukin (IL)-2 and IL-21 dichotomously shape CD8+ T cell differentiation. IL-2 drives terminal differentiation, generating cells that are poorly effective against tumors, whereas IL-21 promotes stem cell memory T cells (TSCM) and antitumor responses. Here we investigated the role of metabolic programming in the developmental differences induced by these cytokines. IL-2 promoted effector-like metabolism and aerobic glycolysis, robustly inducing lactate dehydrogenase (LDH) and lactate production, whereas IL-21 maintained a metabolically quiescent state dependent on oxidative phosphorylation. LDH inhibition rewired IL-2-induced effects, promoting pyruvate entry into the tricarboxylic acid cycle and inhibiting terminal effector and exhaustion programs, including mRNA expression of members of the NR4A family of nuclear receptors, as well as Prdm1 and Xbp1 While deletion of Ldha prevented development of cells with antitumor effector function, transient LDH inhibition enhanced the generation of memory cells capable of triggering robust antitumor responses after adoptive transfer. LDH inhibition did not significantly affect IL-21-induced metabolism but caused major transcriptomic changes, including the suppression of IL-21-induced exhaustion markers LAG3, PD1, 2B4, and TIM3. LDH inhibition combined with IL-21 increased the formation of TSCM cells, resulting in more profound antitumor responses and prolonged host survival. These findings indicate a pivotal role for LDH in modulating cytokine-mediated T cell differentiation and underscore the therapeutic potential of transiently inhibiting LDH during adoptive T cell-based immunotherapy, with an unanticipated cooperative antitumor effect of LDH inhibition and IL-21.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inhibidores Enzimáticos/farmacología , Interleucinas/metabolismo , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Melanoma Experimental/terapia , Células Madre/inmunología , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/trasplante , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Línea Celular Tumoral/trasplante , Humanos , Memoria Inmunológica , Inmunoterapia Adoptiva/métodos , Interleucina-2/inmunología , Interleucina-2/metabolismo , Interleucinas/inmunología , L-Lactato Deshidrogenasa/metabolismo , Melanoma Experimental/inmunología , Ratones , Cultivo Primario de Células , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
Progenitor-like CD8+ T cells mediate long-term immunity to chronic infection and cancer and respond potently to immune checkpoint blockade. These cells share transcriptional regulators with memory precursor cells, including T cell-specific transcription factor 1 (TCF1), but it is unclear whether they adopt distinct programs to adapt to the immunosuppressive environment. By comparing the single-cell transcriptomes and epigenetic profiles of CD8+ T cells responding to acute and chronic viral infections, we found that progenitor-like CD8+ T cells became distinct from memory precursor cells before the peak of the T cell response. We discovered a coexpression gene module containing Tox that exhibited higher transcriptional activity associated with more abundant active histone marks in progenitor-like cells than memory precursor cells. Moreover, thymocyte selection-associated high mobility group box protein TOX (TOX) promoted the persistence of antiviral CD8+ T cells and was required for the programming of progenitor-like CD8+ T cells. Thus, long-term CD8+ T cell immunity to chronic viral infection requires unique transcriptional and epigenetic programs associated with the transcription factor TOX.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Infecciones/etiología , Análisis de la Célula Individual , Animales , Biomarcadores , Inmunoprecipitación de Cromatina , Epigénesis Genética , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de Homeodominio/metabolismo , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Memoria Inmunológica , Infecciones/metabolismo , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Factores de Tiempo , TranscriptomaRESUMEN
T cell senescence and exhaustion are major barriers to successful cancer immunotherapy. Here we show that miR-155 increases CD8+ T cell antitumor function by restraining T cell senescence and functional exhaustion through epigenetic silencing of drivers of terminal differentiation. miR-155 enhances Polycomb repressor complex 2 (PRC2) activity indirectly by promoting the expression of the PRC2-associated factor Phf19 through downregulation of the Akt inhibitor, Ship1. Phf19 orchestrates a transcriptional program extensively shared with miR-155 to restrain T cell senescence and sustain CD8+ T cell antitumor responses. These effects rely on Phf19 histone-binding capacity, which is critical for the recruitment of PRC2 to the target chromatin. These findings establish the miR-155-Phf19-PRC2 as a pivotal axis regulating CD8+ T cell differentiation, thereby paving new ways for potentiating cancer immunotherapy through epigenetic reprogramming of CD8+ T cell fate.
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Linfocitos T CD8-positivos/inmunología , Melanoma Experimental/inmunología , MicroARNs/metabolismo , Neoplasias Cutáneas/inmunología , Factores de Transcripción/metabolismo , Traslado Adoptivo/métodos , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/trasplante , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Senescencia Celular/genética , Senescencia Celular/inmunología , Epigénesis Genética/inmunología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma Experimental/genética , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Complejo Represivo Polycomb 2/inmunología , Complejo Represivo Polycomb 2/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/terapia , Factores de Transcripción/genética , Factores de Transcripción/inmunologíaRESUMEN
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.
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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
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
Several lines of evidence indirectly suggest that antigenic stimulation through the B-cell receptor (BCR) supports chronic lymphocytic leukemia (CLL) development. In addition to self-antigens, a number of microbial antigens have been proposed to contribute to the selection of the immunoglobulins expressed in CLL. How pathogen-specific BCRs drive CLL development remains, however, largely unexplored. Here, we utilized mouse models of CLL pathogenesis to equip B cells with virus-specific BCRs and study the effect of antigen recognition on leukemia growth. Our results show that BCR engagement is absolutely required for CLL development. Unexpectedly, however, neither acute nor chronic exposure to virus-derived antigens influenced leukemia progression. Rather, CLL clones preferentially selected light chains that, when paired with virus-specific heavy chains, conferred B cells the ability to recognize a broad range of autoantigens. Taken together, our results suggest that pathogens may drive CLL pathogenesis by selecting and expanding pathogen-specific B cells that cross-react with one or more self-antigens.
Asunto(s)
Autoantígenos/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Cadenas Ligeras de Inmunoglobulina/metabolismo , Inmunoglobulinas/metabolismo , Molécula 3 de Adhesión Intercelular/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis por Matrices de Proteínas , Proteínas Proto-Oncogénicas/genética , Bazo/citología , Bazo/metabolismo , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/metabolismoRESUMEN
BACKGROUND & AIMS: Besides secreting pro-inflammatory cytokines, chemokines and effector molecules, effector CD8+ T cells that arise upon acute infection with certain viruses have been shown to produce the regulatory cytokine interleukin (IL)-10 and, therefore, contain immunopathology. Whether the same occurs during acute hepatitis B virus (HBV) infection and role that IL-10 might play in liver disease is currently unknown. METHODS: Mouse models of acute HBV pathogenesis, as well as chimpanzees and patients acutely infected with HBV, were used to analyse the role of CD8+ T cell-derived IL-10 in liver immunopathology. RESULTS: Mouse HBV-specific effector CD8+ T cells produce significant amounts of IL-10 upon in vivo antigen encounter. This is corroborated by longitudinal data in a chimpanzee acutely infected with HBV, where serum IL-10 was readily detectable and correlated with intrahepatic CD8+ T cell infiltration and liver disease severity. Unexpectedly, mouse and human CD8+ T cell-derived IL-10 was found to act in an autocrine/paracrine fashion to enhance IL-2 responsiveness, thus preventing antigen-induced HBV-specific effector CD8+ T cell apoptosis. Accordingly, the use of mouse models of HBV pathogenesis revealed that the IL-10 produced by effector CD8+ T cells promoted their own intrahepatic survival and, thus supported, rather than suppressed liver immunopathology. CONCLUSION: Effector CD8+ T cell-derived IL-10 enhances acute liver immunopathology. Altogether, these results extend our understanding of the cell- and tissue-specific role that IL-10 exerts in immune regulation. Lay summary: Interleukin-10 is mostly regarded as an immunosuppressive cytokine. We show here that HBV-specific CD8+ T cells produce IL-10 upon antigen recognition and that this cytokine enhances CD8+ T cell survival. As such, IL-10 paradoxically promotes rather than suppresses liver disease.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Interleucina-10/fisiología , Hígado/inmunología , Enfermedad Aguda , Animales , Apoptosis , Virus de la Hepatitis B/inmunología , Humanos , Interleucina-2/farmacología , Hígado/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Pan troglodytesRESUMEN
Antibodies are critical for protection against viral infections. However, several viruses, such as lymphocytic choriomeningitis virus (LCMV), avoid the induction of early protective antibody responses by poorly understood mechanisms. Here we analyzed the spatiotemporal dynamics of B cell activation to show that, upon subcutaneous infection, LCMV-specific B cells readily relocate to the interfollicular and T cell areas of the draining lymph node where they extensively interact with CD11b+Ly6Chi inflammatory monocytes. These myeloid cells were recruited to lymph nodes draining LCMV infection sites in a type I interferon-, CCR2-dependent fashion and they suppressed antiviral B cell responses by virtue of their ability to produce nitric oxide. Depletion of inflammatory monocytes, inhibition of their lymph node recruitment or impairment of their nitric oxide-producing ability enhanced LCMV-specific B cell survival and led to robust neutralizing antibody production. In conclusion, our results identify inflammatory monocytes as critical gatekeepers that prevent antiviral B cell responses and suggest that certain viruses take advantage of these cells to prolong their persistence within the host.
RESUMEN
Scavenger receptor class B type I (SR-B1) binds pathogen-associated molecular patterns participating in the regulation of the inflammatory reaction but there is no information regarding potential interactions between SR-B1 and the interferon system. Herein, we report that SR-B1 ligands strongly regulate the transcriptional response to interferon α (IFNα) and enhance its antiviral and antitumor activity. This effect was mediated by the activation of TLR2 and TLR4 as it was annulled by the addition of anti-TLR2 or anti-TLR4 blocking antibodies. In vivo, we maximized the antitumor activity of IFNα co-expressing in the liver a SR-B1 ligand and IFNα by adeno-associated viruses. This gene therapy strategy eradicated liver metastases from colon cancer with reduced toxicity. On the other hand, genetic and pharmacological inhibition of SR-B1 blocks the clathrin-dependent interferon receptor recycling pathway with a concomitant reduction in IFNα signaling and bioactivity. This effect can be applied to enhance cancer immunotherapy with oncolytic viruses. Indeed, SR-B1 antagonists facilitate replication of oncolytic viruses amplifying their tumoricidal potential. In conclusion, SR-B1 agonists behave as IFNα enhancers while SR-B1 inhibitors dampen IFNα activity. These results demonstrate that SR-B1 is a suitable pharmacology target to enhance cancer immunotherapy based on IFNα and oncolytic viruses.
RESUMEN
Effector CD8(+) T cells (CD8 TE) play a key role during hepatotropic viral infections. Here, we used advanced imaging in mouse models of hepatitis B virus (HBV) pathogenesis to understand the mechanisms whereby these cells home to the liver, recognize antigens, and deploy effector functions. We show that circulating CD8 TE arrest within liver sinusoids by docking onto platelets previously adhered to sinusoidal hyaluronan via CD44. After the initial arrest, CD8 TE actively crawl along liver sinusoids and probe sub-sinusoidal hepatocytes for the presence of antigens by extending cytoplasmic protrusions through endothelial fenestrae. Hepatocellular antigen recognition triggers effector functions in a diapedesis-independent manner and is inhibited by the processes of sinusoidal defenestration and capillarization that characterize liver fibrosis. These findings reveal the dynamic behavior whereby CD8 TE control hepatotropic pathogens and suggest how liver fibrosis might reduce CD8 TE immune surveillance toward infected or transformed hepatocytes.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Virus de la Hepatitis B/fisiología , Hepatitis B/inmunología , Hígado/inmunología , Monitorización Inmunológica , Animales , Movimiento Celular , Células Endoteliales/metabolismo , Hepatitis B/patología , Hepatocitos/metabolismo , Ácido Hialurónico/metabolismo , Hígado/citología , Cirrosis Hepática , Ratones , Ratones Endogámicos C57BL , Adhesividad Plaquetaria , Organismos Libres de Patógenos EspecíficosRESUMEN
BACKGROUND & AIMS: Current hepatitis B virus (HBV) management is challenging as treatment with nucleos(t)ide analogues needs to be maintained indefinitely and because interferon (IFN)-α therapy is associated with considerable toxicity. Previously, we showed that linking IFNα to apolipoprotein A-I generates a molecule (IA) with distinct antiviral and immunostimulatory activities which lacks the hematological toxicity of IFNα. METHODS: Here, we analyse the antiviral potential of an adeno-associated vector encoding IFNα fused to apolipoprotein A-I (AAV-IA) in comparison to a vector encoding only IFNα (AAV-IFN) in two animal models of chronic hepadnavirus infection. RESULTS: In HBV transgenic mice, we found that both vectors induced marked reductions in serum and liver HBV DNA and in hepatic HBV RNA but AAV-IFN caused lethal pancytopenia. Woodchucks with chronic hepatitis virus (WHV) infection that were treated by intrahepatic injection of vectors encoding the woodchuck sequences (AAV-wIFN or AAV-wIA), experienced only a slight reduction of viremia which was associated with hematological toxicity and high mortality when using AAV-wIFN, while AAV-wIA was well tolerated. However, when we tested AAV-wIA or a control vector encoding woodchuck apolipoprotein A-I (AAV-wApo) in combination with entecavir, we found that AAV-wApo-treated animals exhibited an immediate rebound of viral load upon entecavir withdrawal while, in AAV-wIA-treated woodchucks, viremia and antigenemia remained at low levels for several weeks following entecavir interruption. CONCLUSIONS: Treatment with AAV-IA is safe and elicits antiviral effects in animal models with difficult to treat chronic hepadnavirus infection. AAV-IA in combination with nucleos(t)ide analogues represents a promising approach for the treatment of HBV infection in highly viremic patients.
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Apolipoproteína A-I/metabolismo , ADN Viral/genética , Terapia Genética/métodos , Hepadnaviridae/genética , Hepatitis B Crónica/terapia , Interferón-alfa/uso terapéutico , Hígado/efectos de los fármacos , Animales , Antivirales/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos , Hepatitis B Crónica/genética , Hepatitis B Crónica/virología , Hígado/virología , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Transforming growth factor ß (TGF-ß) is a powerful promoter of cancer progression and a key target for antitumor therapy. As cancer cells exhibit active cholesterol metabolism, high density lipoproteins (HDLs) appear as an attractive delivery system for anticancer TGFß-inhibitory molecules. We constructed a plasmid encoding a potent TGF-ß-blocking peptide (P144) linked to apolipoprotein A-I (ApoA-I) through a flexible linker (pApoLinkerP144). The ApoLinkerP144 sequence was then incorporated into a hepatotropic adeno-associated vector (AAVApoLinkerP144). The aim was to induce hepatocytes to produce HDLs containing a modified ApoA-I capable of blocking TGF-ß. We observed that transduction of the murine liver with pApoLinkerP144 led to the appearance of a fraction of circulating HDL containing the fusion protein. These HDLs were able to attenuate TGF-ß signaling in the liver and to enhance IL-12 -mediated IFN-γ production. Treatment of liver metastasis of MC38 colorectal cancer with AAVApoLinkerP144 resulted in a significant reduction of tumor growth and enhanced expression of IFN-γ and GM-CSF in cancerous tissue. ApoLinkerP144 also delayed MC38 liver metastasis in Rag2-/-IL2rγ-/- immunodeficient mice. This effect was associated with downregulation of TGF-ß target genes essential for metastatic niche conditioning. Finally, in a subset of ret transgenic mice, a model of aggressive spontaneous metastatic melanoma, AAVApoLinkerP144 delayed tumor growth in association with increased CD8+ T cell numbers in regional lymph nodes. In conclusion, modification of HDLs to transport TGF-ß-blocking molecules is a novel and promising approach to inhibit the growth of liver metastases by immunological and non-immunological mechanisms.
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Neoplasias Colorrectales/patología , Lipoproteínas HDL/farmacología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Terapia Molecular Dirigida , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Complejo CD3/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Femenino , Vectores Genéticos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Hepatocitos/metabolismo , Interferón gamma/metabolismo , Hígado/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Metástasis de la Neoplasia/prevención & control , Plásmidos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-ret/genética , Factor de Crecimiento Transformador beta1/antagonistas & inhibidoresRESUMEN
Interleukin (IL)-15 effects on CD8 T and natural killer (NK) lymphocytes hold promise to treat cancer. Fusion proteins have been engineered to provide IL-15 receptor alpha (IL-15Rα) mediated trans-presentation to lymphocytes and extend the plasma half-life of the cytokine. In this study, we report on a triple fusion protein combining apolipoprotein A-I (Apo A-I), IL-15, and IL-15Rα's sushi domain. Apo A-I conveys IL-15 to high-density lipoproteins (HDL), from which the cytokine is trans-presented by the IL-15Rα's sushi domain. Such a construction was tested by hydrodynamic gene transfer to the liver of mice. Lethal toxicity was observed upon injection of 10 µg of the expression plasmid. Mice died from an acute lymphocytic pneumonitis in which T and NK cells dominate a severe inflammatory infiltrate. Importantly, mice devoid of NK cells were not susceptible to such toxicity and mice lacking granzymes A and B also survived the otherwise lethal gene transfer. Lower plasmid doses (<2.5 µg) were tolerated and dramatically increased the numbers of NK and memory CD8 T lymphocytes in the liver, spleen, and lungs, to the point of rescuing the deficiency of such lymphocyte subsets in IL-15Rα(-/-) mice. Doses of plasmid within the therapeutic window successfully treated metastatic tumor models, including B16OVA lung metastasis of melanoma and MC38 colon cancer liver metastasis. Sushi-IL-15-Apo as a recombinant protein was also bioactive in vivo, became conjugated to HDL, and displayed immunotherapeutic effects against metastatic disease.
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Inmunoterapia/métodos , Interleucina-15/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Animales , Apolipoproteína A-I/inmunología , Apolipoproteína A-I/farmacología , Linfocitos T CD8-positivos/inmunología , HDL-Colesterol/inmunología , HDL-Colesterol/farmacología , Técnicas de Transferencia de Gen , Interleucina-15/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plásmidos , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Interferon alpha linked to apolipoprotein A-I has been recently proposed as an improved interferon-based therapy. In the present study, we aimed to develop a computational model to gain further insight into the in vivo behaviour of this new fusion protein. In order to facilitate in vivo evaluation of interferon and the fusion protein without altering their biological properties, green fluorescent protein was incorporated into their structures. Kinetic and dynamic behaviour of both compounds was successfully described after plasmid hydrodynamic administration and in situ synthesis of the studied proteins. Results from the modelling exercise showed that apolipoprotein A-I conferred a modified kinetic behaviour, varying molecule distribution and prolonging half-life without altering liver dynamic performance. However, differences in the gene expression activity were observed at brain level between both compounds. Those differences could be explained by modifications in the dynamic, but also in the biodistribution properties, which would be worth evaluating in future experiments. Therefore, the modelling approach provided a global comprehension of a complex system and allowed us to compare the in vivo behaviour of both compounds and to identify critical aspects that might be important to understand the system better and suggests a need for new model-based experiments.
Asunto(s)
Apolipoproteína A-I/metabolismo , Simulación por Computador , Interferón-alfa/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Encéfalo/metabolismo , Femenino , Células HEK293 , Humanos , Cinética , Ratones , Ratones Endogámicos BALB C , Modelos BiológicosRESUMEN
IFN-α is widely used for the treatment of chronic viral hepatitis and malignancies. However, systemic IFN-α treatment causes severe neuropsychiatric complications in humans, including depression, anxiety, and cognitive impairments. We have previously reported that the fusion protein formed by IFN-α and apolipoprotein A-I (IA) circulates bound to high-density lipoproteins (HDLs) and exhibits liver targeting, increased half-life, enhanced immunostimulatory activity, and reduced cytotoxicity. As the transport of HDLs across the blood-brain barrier is a highly complex and regulated process, in this study, we examine the effects of IA on the brain. Determination of IFN-α in brain and serum after hydrodynamic administration of different doses of a plasmid encoding IFN-α or IA showed that IA penetrated into the brain by a saturable transport mechanism. Thus, at high serum levels of the transgenes, the induction of IFN-sensitive genes and the number of phospho-STAT1(+) cell nuclei in the brain were substantially higher with IFN-α than with IA. This was associated with attenuation of neurodepression in mice given IA, as manifested by shorter immobility time in the tail suspension test. However, when given low doses of rIFN-α or the same antiviral units of HDLs containing IA, the induction of IFN-stimulated genes in the brain was significantly greater with the latter. In conclusion, IA crosses the blood-brain barrier not by diffusion, as is the case of IFN-α, but by a facilitated saturable transport mechanism. Thus, linkage to apolipoprotein A-I may serve to modulate the effects of IFN-α on the CNS.
Asunto(s)
Apolipoproteína A-I/metabolismo , Barrera Hematoencefálica/metabolismo , Interferón-alfa/metabolismo , Proteínas Recombinantes de Fusión/farmacocinética , Animales , Apolipoproteína A-I/genética , Depresión/tratamiento farmacológico , Depresión/etiología , Femenino , Semivida , Suspensión Trasera , Humanos , Inyecciones Intravenosas , Interferón-alfa/genética , Lipoproteínas HDL/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Apolipoprotein A-I (Apo A-I) is a major component of high density lipoproteins (HDL) that transport cholesterol in circulation. We have constructed an expression plasmid encoding a chimeric molecule encompassing interleukin-15 (IL-15) and Apo A-I (pApo-hIL15) that was tested by hydrodynamic injections into mice and was co-administered with a plasmid encoding the sushi domain of IL-15Rα (pSushi) in order to enhance IL-15 trans-presentation and thereby bioactivity. The pharmacokinetics of the Apo A-I chimeric protein were much longer than non-stabilized IL-15 and its bioactivity was enhanced in combination with IL-15Rα Sushi. Importantly, the APO-IL-15 fusion protein was incorporated in part into circulating HDL. Liver gene transfer of these constructs increased NK and memory-phenotype CD8 lymphocyte numbers in peripheral blood, spleen and liver as a result of proliferation documented by CFSE dilution and BrdU incorporation. Moreover, the gene transfer procedure partly rescued the NK and memory T-cell deficiency observed in IL-15Rα(-/-) mice. pApo-hIL15+ pSushi gene transfer to the liver showed a modest therapeutic activity against subcutaneously transplanted MC38 colon carcinoma tumors, that was more evident when tumors were set up as liver metastases. The improved pharmacokinetic profile and the strong biological activity of APO-IL-15 fusion protein holds promise for further development in combination with other immunotherapies.
Asunto(s)
Técnicas de Transferencia de Gen , Terapia Genética , Inmunoterapia , Interleucina-15/genética , Interleucina-15/uso terapéutico , Lipoproteínas HDL/sangre , Hígado/metabolismo , Animales , Apolipoproteína A-I/sangre , Western Blotting , Linfocitos T CD8-positivos/inmunología , Recuento de Células , Línea Celular Tumoral , Proliferación Celular , Humanos , Memoria Inmunológica , Inyecciones Subcutáneas , Interleucina-15/sangre , Subunidad alfa del Receptor de Interleucina-15/química , Subunidad alfa del Receptor de Interleucina-15/metabolismo , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Fenotipo , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , Bazo/metabolismoRESUMEN
Cervical carcinoma is one of the most common cancers in women worldwide. It is well established that chronic infection of the genital tract by various mucosatropic human papillomavirus (HPV) types causes cervical cancer. Cellular immunity to E7 protein from HPV (HPVE7) has been associated with clinical and cytologic resolution of HPV-induced lesions. Thus, we decided to test if targeting of HPVE7 to dendritic cells using a fusion protein containing the extra domain A (EDA) from fibronectin, a natural ligand for TLR4, and HPVE7 (EDA-HPVE7) might be an efficient vaccine for the treatment of cervical carcinoma. We found that EDA-HPVE7 fusion protein was efficiently captured by bone marrow derived dendritic cells in vitro and induced their maturation, with the upregulation of maturation markers and the production of IL-12. Immunization of mice with EDA-HPVE7 fusion protein induced antitumor CD8(+) T cell responses in the absence of additional adjuvants. Repeated intratumoral administration of EDA-HPVE7 in saline was able to cure established TC-1 tumors of 5-7 mm in diameter. More importantly, intravenous injection with EDA-HPVE7 in combination with the TLR ligand polyinosinic-polycytidylic acid (pIC), or with low doses of cyclophosphamide and the TLR9 ligand CpG-B complexed in cationic lipids, were able to eradicate large established TC-1 tumors (1.2 cm in diameter). Thus, therapeutic vaccination with EDA-HPVE7 fusion protein may be effective in the treatment of human cervical carcinoma.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Fibronectinas/inmunología , Proteínas E7 de Papillomavirus/inmunología , Vacunas contra Papillomavirus/inmunología , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/terapia , Adyuvantes Inmunológicos , Animales , Células de la Médula Ósea/inmunología , Linfocitos T CD8-positivos/inmunología , Ciclofosfamida/farmacología , Células Dendríticas/inmunología , Femenino , Fibronectinas/metabolismo , Células HEK293 , Humanos , Interleucina-12/biosíntesis , Ratones , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos/farmacología , Papillomaviridae/inmunología , Proteínas E7 de Papillomavirus/metabolismo , Infecciones por Papillomavirus/inmunología , Poli I-C/farmacología , Proteínas Recombinantes de Fusión/inmunología , Neoplasias del Cuello Uterino/virología , Vacunas Sintéticas/inmunologíaRESUMEN
Apolipoprotein A-I (ApoA-I) is the major protein component of high density lipoprotein (HDL) particles in serum, and participates in the reverse transport of cholesterol from tissues to the liver for excretion. The natural HDL tropism to the liver and cancer cells has been used extensively to target encapsulated drugs. The alteration of the plasmatic isoforms of ApoA-I is a hallmark of chronic hepatitis and hepatocarcinoma in mice and humans. Woodchucks infected with the woodchuck hepatitis virus (WHV) represent the best animal model for the study of chronic viral hepatitis B and viral induced hepatocarcinoma (HCC). WHV-infected woodchuck represents a clinically relevant animal model under which new treatment strategies can be evaluated and optimized. Therapeutic efficacy in this model is likely to be translated into a successful therapy for patients infected with HBV. The present study describes, for the first time, the cloning and characterization of woodchuck ApoA-I. The open reading frame (ORF) of the woodchuck ApoA-I is 795 bp long, coding for 264 amino acids. Unexpectedly, phylogenetic analysis revealed that the closest sequences are those of human and macaque. Woodchuck HDLs were isolated successfully from sera by density gradient ultracentrifugation. A commercial antibody that recognized the woodchuck ApoA-I was also identified. Finally, taking advantage of the techniques and tools developed in this study, two potential applications of woodchuck HDLs are illustrated: drug delivery to a woodchuck hepatocarcinoma cell line and the use of isoelectrofocusing to identify ApoA-I isoforms.