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In this article, I aim to give some pieces of career advise for young immunologists based on my own experiences.
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Alergia e Inmunología , Selección de Profesión , Humanos , Alergia e Inmunología/historiaRESUMEN
How the age-associated decline of immune function leads to increased cancer incidence is poorly understood. Here, we have characterised the cellular composition of the γδ T-cell pool in peripheral lymph nodes (pLNs) upon ageing. We find that ageing has minimal cell-intrinsic effects on function and global gene expression of γδ T cells, and γδTCR diversity remains stable. However, ageing alters TCRδ chain usage and clonal structure of γδ T-cell subsets. Importantly, IL-17-producing γδ17 T cells dominate the γδ T-cell pool of aged mice-mainly due to the selective expansion of Vγ6+ γδ17 T cells and augmented γδ17 polarisation of Vγ4+ T cells. Expansion of the γδ17 T-cell compartment is mediated by increased IL-7 expression in the T-cell zone of old mice. In a Lewis lung cancer model, pro-tumourigenic Vγ6+ γδ17 T cells are exclusively activated in the tumour-draining LN and their infiltration into the tumour correlates with increased tumour size in aged mice. Thus, upon ageing, substantial compositional changes in γδ T-cell pool in the pLN lead to an unbalanced γδ T-cell response in the tumour that is associated with accelerated tumour growth.
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Envejecimiento/genética , Carcinoma Pulmonar de Lewis/genética , Regulación Neoplásica de la Expresión Génica , Interleucina-7/genética , Ganglios Linfáticos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Subgrupos de Linfocitos T/inmunología , Envejecimiento/inmunología , Animales , Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/patología , Diferenciación Celular , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Inmunofenotipificación , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-7/inmunología , Ganglios Linfáticos/patología , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T gamma-delta/clasificación , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Transducción de Señal , Subgrupos de Linfocitos T/clasificación , Subgrupos de Linfocitos T/patología , Carga Tumoral/genética , Carga Tumoral/inmunologíaRESUMEN
The repertoire of peptides displayed at the cell surface by MHC I molecules is shaped by two intracellular peptide editors, tapasin and TAPBPR. While cell-free assays have proven extremely useful in identifying the function of both of these proteins, here we explored whether a more physiological system could be developed to assess TAPBPR-mediated peptide editing on MHC I. We reveal that membrane-associated TAPBPR targeted to the plasma membrane retains its ability to function as a peptide editor and efficiently catalyzes peptide exchange on surface-expressed MHC I molecules. Additionally, we show that soluble TAPBPR, consisting of the luminal domain alone, added to intact cells, also functions as an effective peptide editor on surface MHC I molecules. Thus, we have established two systems in which TAPBPR-mediated peptide exchange on MHC class I can be interrogated. Furthermore, we could use both plasma membrane-targeted and exogenous soluble TAPBPR to display immunogenic peptides on surface MHC I molecules and consequently induce T cell receptor engagement, IFN-γ secretion, and T cell-mediated killing of target cells. Thus, we have developed an efficient way to by-pass the natural antigen presentation pathway of cells and load immunogenic peptides of choice onto cells. Our findings highlight a potential therapeutic use for TAPBPR in increasing the immunogenicity of tumors in the future.
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Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulinas/inmunología , Proteínas de la Membrana/inmunología , Péptidos/inmunología , Animales , Células HeLa , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunidad Celular , Inmunoglobulinas/genética , Interferón gamma/genética , Interferón gamma/inmunología , Células MCF-7 , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Péptidos/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunologíaRESUMEN
During the primary response, the commitment of the CD8(+) T cell to Blimp-1 expression and the terminal differentiation that Blimp-1 induces must be timed so as not to impair the process of clonal expansion. We determined whether the Hippo pathway, which links cell-cell contact to differentiation in other cell lineages, controls Blimp-1 expression. Activating the CD8(+) T cell with antigen and IL-2 causes expression of the core Hippo pathway components, including the pivotal transcriptional cofactor Yap. Contact between activated CD8(+) T cells induces Hippo pathway-mediated Yap degradation and Blimp-1 expression; a Hippo-resistant, stable form of Yap suppresses Blimp-1 expression. Cytotoxic T lymphocyte antigen 4 (CTLA-4) and CD80 comprise the receptor-ligand pair that mediates contact-dependent Hippo pathway activation. In vivo, CD8(+) T cells expressing Hippo resistant-Yap or lacking CTLA-4 have diminished expression of the senescence marker, KLRG1, during a viral infection. The CTLA-4/Hippo pathway/Blimp-1 system may couple terminal differentiation of CD8(+) T cell with the magnitude of clonal expansion.
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Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4/metabolismo , Activación de Linfocitos/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/inmunología , Factores de Transcripción/metabolismo , Animales , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Linfocitos T CD8-positivos/citología , Diferenciación Celular/inmunología , Membrana Celular/metabolismo , Activación Enzimática , Ligandos , Ratones , Factor 1 de Unión al Dominio 1 de Regulación PositivaRESUMEN
CD4+ T cells are critical in orchestrating immune responses against various pathogens and cancer but can also be drivers of autoimmune disease, allergy and pro-tumour responses. Naïve CD4+ T cells polarise into specialised T helper cell subsets with unique effector functions. While the guiding transcription factors and effector molecules of the T helper cell lineages are well understood, the signalling pathways orchestrating the intricate T helper cell polarisation programmes remain poorly understood. Here we review an emerging role of Hedgehog signalling - a classical morphogen signalling pathway - in T helper cell polarisation. Importantly, the Hedgehog pathway is pharmacologically highly tractable and existing clinically-approved Hedgehog inhibitors may prove useful therapeutic modulators of T helper cell-driven immune responses.
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Linfocitos T CD4-Positivos , Proteínas Hedgehog , Transducción de Señal , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular , Proteínas Hedgehog/metabolismo , Linfocitos T Colaboradores-Inductores , Factores de Transcripción/metabolismo , Humanos , AnimalesRESUMEN
Imaging reporter genes are indispensable for visualising biological processes in living subjects, particularly in cancer research where they have been used to observe tumour development, cancer cell dissemination, and treatment response. Engineering reporter genes into the germline frequently involves single imaging modality reporters operating over limited spatial scales. To address these limitations, we developed an inducible triple-reporter mouse model (Rosa26LSL - NRL) that integrates reporters for complementary imaging modalities, flfluorescence, bioluminescence and positron emission tomography (PET), along with inducible Cre-lox functionality for precise spatiotemporal control of reporter expression. We demonstrated robust reporter inducibility across various tissues in the Rosa26LSL - NRL mouse, facilitating effective tracking and characterisation of tumours in liver and lung cancer mouse models. We precisely pinpointed tumour location using multimodal whole-body imaging which guided in situ lung microscopy to visualise cell-cell interactions within the tumour microenvironment. The triple-reporter system establishes a robust new platform technology for multi-scale investigation of biological processes within whole animals, enabling tissue-specific and sensitive cell tracking, spanning from the whole-body to cellular scales.
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We have developed and validated a highly specific, versatile antibody to the extracellular domain of human LGR5 (α-LGR5). α-LGR5 detects LGR5 overexpression in >90% of colorectal cancer (CRC), hepatocellular carcinoma (HCC) and pre-B-ALL tumour cells and was used to generate an Antibody-Drug Conjugate (α-LGR5-ADC), Bispecific T-cell Engager (α-LGR5-BiTE) and Chimeric Antigen Receptor (α-LGR5-CAR). α-LGR5-ADC was the most effective modality for targeting LGR5+ cancer cells in vitro and demonstrated potent anti-tumour efficacy in a murine model of human NALM6 pre-B-ALL driving tumour attrition to less than 1% of control treatment. α-LGR5-BiTE treatment was less effective in the pre-B-ALL cancer model yet promoted a twofold reduction in tumour burden. α-LGR5-CAR-T cells also showed specific and potent LGR5+ cancer cell killing in vitro and effective tumour targeting with a fourfold decrease in pre-B-ALL tumour burden relative to controls. Taken together, we show that α-LGR5 can not only be used as a research tool and a biomarker but also provides a versatile building block for a highly effective immune therapeutic portfolio targeting a range of LGR5-expressing cancer cells.
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Th17 cells are key drivers of autoimmune disease. However, the signaling pathways regulating Th17 polarization are poorly understood. Hedgehog signaling regulates cell fate decisions during embryogenesis and adult tissue patterning. Here we find that cell-autonomous Hedgehog signaling, independent of exogenous ligands, selectively drives the polarization of Th17 cells but not other T helper cell subsets. We show that endogenous Hedgehog ligand, Ihh, signals to activate both canonical and non-canonical Hedgehog pathways through Gli3 and AMPK. We demonstrate that Hedgehog pathway inhibition with either the clinically-approved small molecule inhibitor vismodegib or genetic ablation of Ihh in CD4+ T cells greatly diminishes disease severity in two mouse models of intestinal inflammation. We confirm that Hedgehog pathway expression is upregulated in tissue from human ulcerative colitis patients and correlates with Th17 marker expression. This work implicates Hedgehog signaling in Th17 polarization and intestinal immunopathology and indicates the potential therapeutic use of Hedgehog inhibitors in the treatment of inflammatory bowel disease.
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Colitis Ulcerosa , Células Th17 , Adulto , Animales , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Transducción de Señal , VirulenciaRESUMEN
In vitro assays that evaluate CD8+ T cell-mediated cytotoxicity are important to aid in the development of novel therapeutic approaches to enhance anti-tumor immune responses. Here, we describe a novel cytotoxicity co-culture assay that circumvents the problem of highly variable allogeneic responses and obviates the constraints of HLA-restriction between effector and target cells. We show that this assay can be easily applied to a panel of tumor cell lines to provide additional insights into intrinsic drivers of sensitivity/resistance to T cell-mediated killing, and to evaluate the impact of targeted therapies on both tumor and T cell compartments.
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Macroautophagy/autophagy is an evolutionarily conserved catabolic pathway whose modulation has been linked to diverse disease states, including age-associated disorders. Conventional and conditional whole-body knockout mouse models of key autophagy genes display perinatal death and lethal neurotoxicity, respectively, limiting their applications for in vivo studies. Here, we have developed an inducible shRNA mouse model targeting Atg5, allowing us to dynamically inhibit autophagy in vivo, termed ATG5i mice. The lack of brain-associated shRNA expression in this model circumvents the lethal phenotypes associated with complete autophagy knockouts. We show that ATG5i mice recapitulate many of the previously described phenotypes of tissue-specific knockouts. While restoration of autophagy in the liver rescues hepatomegaly and other pathologies associated with autophagy deficiency, this coincides with the development of hepatic fibrosis. These results highlight the need to consider the potential side effects of systemic anti-autophagy therapies.
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Proteína 5 Relacionada con la Autofagia/metabolismo , Autofagia , ARN Interferente Pequeño/metabolismo , Animales , Animales Recién Nacidos , Proteína 5 Relacionada con la Autofagia/genética , Regulación hacia Abajo/genética , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Modelos Animales , Fenotipo , Factores de TiempoRESUMEN
Primary cilia are microtubule-based organelles that detect mechanical and chemical stimuli. Although cilia house a number of oncogenic molecules (including Smoothened, KRAS, EGFR, and PDGFR), their precise role in cancer remains unclear. We have interrogated the role of cilia in acquired and de novo resistance to a variety of kinase inhibitors, and found that, in several examples, resistant cells are distinctly characterized by an increase in the number and/or length of cilia with altered structural features. Changes in ciliation seem to be linked to differences in the molecular composition of cilia and result in enhanced Hedgehog pathway activation. Notably, manipulating cilia length via Kif7 knockdown is sufficient to confer drug resistance in drug-sensitive cells. Conversely, targeting of cilia length or integrity through genetic and pharmacological approaches overcomes kinase inhibitor resistance. Our work establishes a role for ciliogenesis and cilia length in promoting cancer drug resistance and has significant translational implications.
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Cilios/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias/patología , Inhibidores de Proteínas Quinasas/farmacología , Línea Celular Tumoral , Cilios/efectos de los fármacos , Proteínas Hedgehog/metabolismo , Humanos , Modelos Biológicos , Organogénesis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Aging is characterized by progressive loss of physiological and cellular functions, but the molecular basis of this decline remains unclear. We explored how aging affects transcriptional dynamics using single-cell RNA sequencing of unstimulated and stimulated naïve and effector memory CD4+ T cells from young and old mice from two divergent species. In young animals, immunological activation drives a conserved transcriptomic switch, resulting in tightly controlled gene expression characterized by a strong up-regulation of a core activation program, coupled with a decrease in cell-to-cell variability. Aging perturbed the activation of this core program and increased expression heterogeneity across populations of cells in both species. These discoveries suggest that increased cell-to-cell transcriptional variability will be a hallmark feature of aging across most, if not all, mammalian tissues.
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Envejecimiento/genética , Envejecimiento/inmunología , Linfocitos T CD4-Positivos/inmunología , Memoria Inmunológica/genética , Transcriptoma , Animales , Senescencia Celular/genética , Senescencia Celular/inmunología , Variación Genética , Activación de Linfocitos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula IndividualRESUMEN
Cytotoxic T lymphocytes (CTLs) kill virus-infected and tumour cells with remarkable specificity. Upon recognition, CTLs form a cytolytic immune synapse with their target cell, and marked reorganization of both the actin and the microtubule cytoskeletons brings the centrosome up to the plasma membrane to the point of T cell receptor signalling. Secretory granules move towards the centrosome and are delivered to this focal point of secretion. Such centrosomal docking at the plasma membrane also occurs during ciliogenesis; indeed, striking similarities exist between the cytolytic synapse and the primary cilium that throw light on the possible origins of immune synapses.
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Citotoxicidad Inmunológica/inmunología , Sinapsis Inmunológicas/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Humanos , Sinapsis Inmunológicas/ultraestructura , Linfocitos T Citotóxicos/ultraestructuraRESUMEN
The centrosome is essential for cytotoxic T lymphocyte (CTL) function, contacting the plasma membrane and directing cytotoxic granules for secretion at the immunological synapse. Centrosome docking at the plasma membrane also occurs during cilia formation. The primary cilium, formed in nonhematopoietic cells, is essential for vertebrate Hedgehog (Hh) signaling. Lymphocytes do not form primary cilia, but we found and describe here that Hh signaling played an important role in CTL killing. T cell receptor activation, which "prearms" CTLs with cytotoxic granules, also initiated Hh signaling. Hh pathway activation occurred intracellularly and triggered Rac1 synthesis. These events "prearmed" CTLs for action by promoting the actin remodeling required for centrosome polarization and granule release. Thus, Hh signaling plays a role in CTL function, and the immunological synapse may represent a modified cilium.
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Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica , Proteínas Hedgehog/metabolismo , Sinapsis Inmunológicas , Transducción de Señal , Linfocitos T Citotóxicos/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Polaridad Celular , Células Cultivadas , Centrosoma/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Inmunológicos , Neuropéptidos/genética , Neuropéptidos/metabolismo , Receptores Patched , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Linfocitos T Citotóxicos/metabolismo , Proteína con Dedos de Zinc GLI1 , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Docking of the centrosome at the plasma membrane directs lytic granules to the immunological synapse. To identify signals controlling centrosome docking at the synapse, we have studied cytotoxic T lymphocytes (CTLs) in which expression of the T cell receptor-activated tyrosine kinase Lck is ablated. In the absence of Lck, the centrosome is able to translocate around the nucleus toward the immunological synapse but is unable to dock at the plasma membrane. Lytic granules fail to polarize and release their contents, and target cells are not killed. In CTLs deficient in both Lck and the related tyrosine kinase Fyn, centrosome translocation is impaired, and the centrosome remains on the distal side of the nucleus relative to the synapse. These results show that repositioning of the centrosome in CTLs involves at least two distinct steps, with Lck signaling required for the centrosome to dock at the plasma membrane.