RESUMEN
Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody targeting of the receptor CSF1R, which more broadly eliminates tumor-associated macrophages, reversed tumor growth inhibition in Qpctl-/- mice and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.
Asunto(s)
Aminoaciltransferasas , Linfocitos T CD8-positivos , Quimiocinas , Neoplasias , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Animales , Linfocitos T CD8-positivos/patología , Quimiocinas/metabolismo , Inmunoterapia , Infiltración Leucémica , Ratones , Ratones Noqueados , Monocitos , Neoplasias/inmunologíaRESUMEN
Antigen cross presentation, whereby exogenous antigens are presented by MHC class I molecules to CD8+ T cells, is essential for generating adaptive immunity to pathogens and tumor cells. Following endocytosis, it is widely understood that protein antigens must be transferred from endosomes to the cytosol where they are subject to ubiquitination and proteasome degradation prior to being translocated into the endoplasmic reticulum (ER), or possibly endosomes, via the TAP1/TAP2 complex. Revealing how antigens egress from endocytic organelles (endosome-to-cytosol transfer, ECT), however, has proved vexing. Here, we used two independent screens to identify the hydrogen peroxide-transporting channel aquaporin-3 (AQP3) as a regulator of ECT. AQP3 overexpression increased ECT, whereas AQP3 knockout or knockdown decreased ECT. Mechanistically, AQP3 appears to be important for hydrogen peroxide entry into the endosomal lumen where it affects lipid peroxidation and subsequent antigen release. AQP3-mediated regulation of ECT was functionally significant, as AQP3 modulation had a direct impact on the efficiency of antigen cross presentation in vitro. Finally, AQP3-/- mice exhibited a reduced ability to mount an anti-viral response and cross present exogenous extended peptide. Together, these results indicate that the AQP3-mediated transport of hydrogen peroxide can regulate endosomal lipid peroxidation and suggest that compromised membrane integrity and coordinated release of endosomal cargo is a likely mechanism for ECT.
Asunto(s)
Acuaporina 3/metabolismo , Citosol/metabolismo , Endosomas/metabolismo , Animales , Presentación de Antígeno , Acuaporina 3/genética , Transporte Biológico , Células Cultivadas , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Peroxidación de Lípido , RatonesRESUMEN
Post-translational modification of chemokines mediated by the dipeptidyl peptidase DPP4 (CD26) has been shown to negatively regulate lymphocyte trafficking, and its inhibition enhances T cell migration and tumor immunity by preserving functional chemokine CXCL10. By extending those initial findings to pre-clinical models of hepatocellular carcinoma and breast cancer, we discovered a distinct mechanism by which inhibition of DPP4 improves anti-tumor responses. Administration of the DPP4 inhibitor sitagliptin resulted in higher concentrations of the chemokine CCL11 and increased migration of eosinophils into solid tumors. Enhanced tumor control was preserved in mice lacking lymphocytes and was ablated after depletion of eosinophils or treatment with degranulation inhibitors. We further demonstrated that tumor-cell expression of the alarmin IL-33 was necessary and sufficient for eosinophil-mediated anti-tumor responses and that this mechanism contributed to the efficacy of checkpoint-inhibitor therapy. These findings provide insight into IL-33- and eosinophil-mediated tumor control, revealed when endogenous mechanisms of DPP4 immunoregulation are inhibited.
Asunto(s)
Dipeptidil Peptidasa 4/inmunología , Eosinófilos/inmunología , Interleucina-33/inmunología , Neoplasias Experimentales/inmunología , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Quimiocina CCL11/inmunología , Quimiocina CCL11/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Modelos Animales de Enfermedad , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Humanos , Interleucina-33/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/prevención & control , Fosfato de Sitagliptina/farmacologíaRESUMEN
The preimmune repertoire consists of mature T lymphocytes that have not yet been stimulated in the periphery. Memory phenotype (MP) cells have been reported as part of the preimmune repertoire (i.e., T cells bearing memory markers despite lack of engagement with cognate Ag); however, little is known about their trafficking and function. In this study, we hypothesized that MP cells, naive to TCR stimulation, constitute a transient population that traffics to tissues during development. Using mutant and transgenic animals with a monospecific TCR, we discovered increased numbers of MP CD8+ T cells circulating in nonimmunized Cxcr3-/- and Cxcl10-/- mice compared with wild-type animals. Phenotypic differences included decreased numbers of preimmune MP Ag-specific T cells in the skin and thymus and a distinct pattern of activation upon TCR engagement. Our results show for the first time, to our knowledge, an important role for CXCR3 and CXCL10 in the tissue distribution of preimmune MP cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Quimiocina CXCL10/metabolismo , Receptores CXCR3/metabolismo , Animales , Células Cultivadas , Quimiocina CXCL10/genética , Quimiocina CXCL9/genética , Quimiocina CXCL9/metabolismo , Memoria Inmunológica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores CXCR3/genéticaRESUMEN
The macroautophagy machinery has been implicated in MHC class II restricted antigen presentation. Here, we report that this machinery assists in the internalization of MHC class I molecules. In the absence of the autophagy factors Atg5 and Atg7, MHC class I surface levels are elevated due to decreased endocytosis and degradation. Internalization of MHC class I molecules occurs less efficiently if AAK1 cannot be recruited via Atg8/LC3B. In the absence of Atg-dependent MHC class I internalization, dendritic cells stimulate CD8(+) T cell responses more efficiently in vitro and in vivo. During viral infections, lack of Atg5 results in enhanced influenza- and LCMV-specific CD8(+) T cell responses in vivo. Elevated influenza-specific CD8(+) T cell responses are associated with better immune control of this infection. Thus, the macroautophagy machinery orchestrates T cell immunity by supporting MHC class II but compromises MHC class I restricted antigen presentation.
Asunto(s)
Proteína 5 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/genética , Autofagia/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Animales , Presentación de Antígeno/inmunología , Células Cultivadas , Endocitosis/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Activación de Linfocitos/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Dying cells initiate adaptive immunity by providing both antigens and inflammatory stimuli for dendritic cells, which in turn activate CD8(+) T cells through a process called antigen cross-priming. To define how different forms of programmed cell death influence immunity, we established models of necroptosis and apoptosis, in which dying cells are generated by receptor-interacting protein kinase-3 and caspase-8 dimerization, respectively. We found that the release of inflammatory mediators, such as damage-associated molecular patterns, by dying cells was not sufficient for CD8(+) T cell cross-priming. Instead, robust cross-priming required receptor-interacting protein kinase-1 (RIPK1) signaling and nuclear factor κB (NF-κB)-induced transcription within dying cells. Decoupling NF-κB signaling from necroptosis or inflammatory apoptosis reduced priming efficiency and tumor immunity. Our results reveal that coordinated inflammatory and cell death signaling pathways within dying cells orchestrate adaptive immunity.
Asunto(s)
Apoptosis/inmunología , Linfocitos T CD8-positivos/inmunología , FN-kappa B/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Caspasa 8/metabolismo , Supervivencia Celular , Reactividad Cruzada , Células Dendríticas/inmunología , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Transducción de SeñalRESUMEN
The success of antitumor immune responses depends on the infiltration of solid tumors by effector T cells, a process guided by chemokines. Here we show that in vivo post-translational processing of chemokines by dipeptidylpeptidase 4 (DPP4, also known as CD26) limits lymphocyte migration to sites of inflammation and tumors. Inhibition of DPP4 enzymatic activity enhanced tumor rejection by preserving biologically active CXCL10 and increasing trafficking into the tumor by lymphocytes expressing the counter-receptor CXCR3. Furthermore, DPP4 inhibition improved adjuvant-based immunotherapy, adoptive T cell transfer and checkpoint blockade. These findings provide direct in vivo evidence for control of lymphocyte trafficking via CXCL10 cleavage and support the use of DPP4 inhibitors for stabilizing biologically active forms of chemokines as a strategy to enhance tumor immunotherapy.
Asunto(s)
Dipeptidil Peptidasa 4/inmunología , Inmunoterapia/métodos , Linfocitos/inmunología , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Traslado Adoptivo , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Quimiocina CXCL10/inmunología , Quimiocina CXCL10/metabolismo , Quimiocinas/inmunología , Quimiocinas/metabolismo , Dipeptidil Peptidasa 4/genética , Dipeptidil Peptidasa 4/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Femenino , Citometría de Flujo , Linfocitos/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neoplasias Experimentales/genética , Pirazinas/farmacología , Receptores CXCR3/inmunología , Receptores CXCR3/metabolismo , Fosfato de Sitagliptina , Triazoles/farmacologíaRESUMEN
Cellular zinc homeostasis ensures that the intracellular concentration of this element is kept within limits that enable its participation in critical physiological processes without exerting toxic effects. We report here the identification and characterization of the first mediator of zinc homeostasis in Leishmania infantum, LiZIP3, a member of the ZIP family of divalent metal-ion transporters. The zinc transporter activity of LiZIP3 was first disclosed by its capacity to rescue the growth of Saccharomyces cerevisiae strains deficient in zinc acquisition. Subsequent expression of LiZIP3 in Xenopus laevis oocytes was shown to stimulate the uptake of a broad range of metal ions, among which Zn(2+) was the preferred LiZIP3 substrate (K0.5 ≈ 0.1 µM). Evidence that LiZIP3 functions as a zinc importer in L. infantum came from the observations that the protein locates to the cell membrane and that its overexpression leads to augmented zinc internalization. Importantly, expression and cell-surface location of LiZIP3 are lost when parasites face high zinc bioavailability. LiZIP3 decline in response to zinc is regulated at the mRNA level in a process involving (a) short-lived protein(s). Collectively, our data reveal that LiZIP3 enables L. infantum to acquire zinc in a highly regulated manner, hence contributing to zinc homeostasis.
Asunto(s)
Proteínas Portadoras/metabolismo , Leishmania infantum/metabolismo , Zinc/metabolismo , Animales , Proteínas Portadoras/genética , Perfilación de la Expresión Génica , Prueba de Complementación Genética , Leishmania infantum/enzimología , Leishmania infantum/genética , Oocitos/enzimología , Oocitos/metabolismo , Proteínas Protozoarias/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , Xenopus laevisRESUMEN
Human CD34(+) progenitor-derived Langerhans-type dendritic cells (LCs) are more potent stimulators of T-cell immunity against tumor and viral antigens in vitro than are monocyte-derived DCs (moDCs). The exact mechanisms have remained elusive until now, however. LCs synthesize the highest amounts of IL-15R-α mRNA and protein, which binds IL-15 for presentation to responder lymphocytes, thereby signaling the phosphorylation of signal transducer and activator of transcription 5 (pSTAT5). LCs electroporated with Wilms tumor 1 (WT1) mRNA achieve sufficiently sustained presentation of antigenic peptides, which together with IL-15R-α/IL-15, break tolerance against WT1 by stimulating robust autologous, WT1-specific cytolytic T-lymphocytes (CTLs). These CTLs develop from healthy persons after only 7 days' stimulation without exogenous cytokines and lyse MHC-restricted tumor targets, which include primary WT1(+) leukemic blasts. In contrast, moDCs require exogenous rhuIL-15 to phosphorylate STAT5 and attain stimulatory capacity comparable to LCs. LCs therefore provide a more potent costimulatory cytokine milieu for T-cell activation than do moDCs, thus accounting for their superior stimulation of MHC-restricted Ag-specific CTLs without need for exogenous cytokines. These data support the use of mRNA-electroporated LCs, or moDCs supplemented with exogenous rhuIL-15, as vaccines for cancer immunotherapy to break tolerance against self-differentiation antigens shared by tumors.
Asunto(s)
Presentación de Antígeno , Tolerancia Inmunológica , Interleucina-15/inmunología , Células de Langerhans/inmunología , Receptores de Interleucina-15/inmunología , Factor de Transcripción STAT5/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas WT1/inmunología , Crisis Blástica/genética , Crisis Blástica/inmunología , Crisis Blástica/patología , Crisis Blástica/terapia , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/farmacología , Femenino , Humanos , Interleucina-15/farmacología , Células de Langerhans/patología , Leucemia/genética , Leucemia/inmunología , Leucemia/patología , Leucemia/terapia , Activación de Linfocitos/efectos de los fármacos , Masculino , Receptores de Interleucina-15/genética , Factor de Transcripción STAT5/genética , Linfocitos T Citotóxicos/patología , Proteínas WT1/genéticaRESUMEN
Human mature dendritic cells (DCs) can efficiently stimulate natural killer (NK)-cell responses without being targeted by their cytotoxicity. To understand this important regulatory crosstalk, we characterized the development of the immunologic synapse between mature DCs and resting NK cells. Conjugates between these 2 innate leukocyte populations formed rapidly, persisted for prolonged time periods and matured with DC-derived f-actin polymerization at the synapse. Polarization of IL-12 and IL-12R to the synapse coincided with f-actin polymerization, while other activating and inhibitory molecules were enriched at the interface between DCs and NK cells earlier. Functional assays revealed that inhibition of f-actin polymerization in mature synapses led to an increase of IFN-γ secretion and cytotoxicity by NK cells. This elevated NK-cell reactivity resulted from decreased inhibitory signaling in the absence of MHC class I polarization at the interface, which was observed on inhibition of f-actin polymerization in DCs. Thus, inhibitory signaling is stabilized by f-actin at the synapse between mature DCs and resting NK cells.
Asunto(s)
Citoesqueleto/inmunología , Células Dendríticas/inmunología , Sinapsis Inmunológicas/inmunología , Células Asesinas Naturales/inmunología , Actinas/inmunología , Actinas/metabolismo , Comunicación Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Citoesqueleto/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/ultraestructura , Citometría de Flujo , Humanos , Sinapsis Inmunológicas/metabolismo , Interleucina-12/inmunología , Interleucina-12/metabolismo , Subunidad alfa del Receptor de Interleucina-15/inmunología , Subunidad alfa del Receptor de Interleucina-15/metabolismo , Células K562 , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/ultraestructura , Microscopía Confocal , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Centro Organizador de los Microtúbulos/inmunología , Centro Organizador de los Microtúbulos/metabolismo , Interferencia de ARN , Receptores de Interleucina-12/inmunología , Receptores de Interleucina-12/metabolismo , Receptores KIR/inmunología , Receptores KIR/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/inmunología , Proteína del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Natural killer (NK) cells have originally been identified by their spontaneous cytolytic potential against tumor cells, which, however, might result from pre-activation due to prior pathogen exposure. Resting NK cells, on the contrary, require activation by bystander antigen-presenting cells to reach their full functional competence. In this review, we will summarize studies on how dendritic cells (DCs), the most potent type of antigen-presenting cell, communicate with human NK cells to activate them in secondary lymphoid organs and to integrate signals from activated NK cells at sites of inflammation for their own maturation. Furthermore, we will review aspects of the immunological synapse, which mediates this cross-talk. These studies provide the mechanistic understanding of how mature DCs can activate NK cells and survive to go on for the activation of adaptive immunity. This feature of DCs, to activate different waves of immune responses, could be harnessed for immunotherapies, including vaccinations.
Asunto(s)
Células Dendríticas/metabolismo , Células Asesinas Naturales/metabolismo , Activación de Linfocitos , Transducción de Señal , Células Dendríticas/citología , Células Dendríticas/inmunología , Humanos , Sinapsis Inmunológicas/inmunología , Sinapsis Inmunológicas/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunologíaRESUMEN
Microglia are resident macrophage-like APCs of the CNS. To avoid escalation of inflammatory processes and bystander damage within the CNS, microglia-driven inflammatory responses need to be tightly regulated and both spatially and temporally restricted. Following traumatic, infectious, and autoimmune-mediated brain injury, NK cells have been found in the CNS, but the functional significance of NK cell recruitment and their mechanisms of action during brain inflammation are not well understood. In this study, we investigated whether and by which mechanisms human NK cells might edit resting and activated human microglial cells via killing in vitro. IL-2-activated NK cells efficiently killed both resting allogeneic and autologous microglia in a cell-contact-dependent manner. Activated NK cells rapidly formed synapses with human microglial cells in which perforin had been polarized to the cellular interface. Ab-mediated NKG2D and NKp46 blockade completely prevented the killing of human microglia by activated NK cells. Up-regulation of MHC class I surface expression by TLR4 stimulation protected microglia from NK cell-mediated killing, whereas MHC class I blockade enhanced cytotoxic NK cell activity. These data suggest that brain-infiltrating NK cells might restrict innate and adaptive immune responses within the human CNS via elimination of resting microglia.