RESUMEN
Allogeneic chimeric antigen receptor (CAR)-expressing T cells offer many advantages over autologous therapies, but their benefits are curtailed by graft-versus-host disease and elimination by recipient immune cells. Moreover, just as with autologous therapies, allogeneic CAR T cells are susceptible to activation-induced cell death (AICD) caused by chronic antigen exposure (CAE). Granzyme B- and Fas/Fas ligand-initiated caspase-mediated apoptoses are key mechanisms of T-cell death caused by T/NK cell-mediated allorejection or CAE. We explored a protective strategy of engineering CAR T cells to overexpress variants of the Granzyme B-specific serine protease inhibitor SERPINB9 (SB9) to improve allogeneic T-cell persistence and antitumor efficacy. We showed that the overexpression of an SB9 variant with broadened caspase specificity, SB9(CAS), not only significantly reduced rejection of allogeneic CAR T cells but also increased their resistance to AICD and enabled them to thrive better under CAE, thus improving allogeneic T-cell persistence and antitumor activity in vitro and in vivo. In addition, although SB9(CAS) overexpression improved the efficacy of allogeneic CAR T-cell therapy by conferring protection to cell death, we did not observe any autonomous growth, and the engineered CAR T cells were still susceptible to an inducible suicide switch. Hence, SB9(CAS) overexpression is a promising strategy that can strengthen current development of cell therapies, broadening their applications to address unmet medical needs.
Asunto(s)
Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos , Serpinas , Linfocitos T , Animales , Humanos , Serpinas/genética , Serpinas/metabolismo , Inmunoterapia Adoptiva/métodos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ratones , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Línea Celular TumoralRESUMEN
Themis is important in regulating positive selection of thymocytes during T cell development, but its role in peripheral T cells is less understood. Here, we investigated T cell activation and its sequelae using a tamoxifen-mediated, acute Themis deletion mouse model. We find that proliferation, effector functions including anti-tumor killing, and up-regulation of energy metabolism are severely compromised. This study reveals the phenomenon of peripheral adaptation to loss of Themis, by demonstrating direct TCR-induced defects after acute deletion of Themis that were not evident in peripheral T cells chronically deprived of Themis in dLck-Cre deletion model. Peripheral adaptation to long-term loss was compared using chronic versus acute tamoxifen-mediated deletion and with the (chronic) dLck-Cre deletion model. We found that upon chronic tamoxifen-mediated Themis deletion, there was modulation in the gene expression profile for both TCR and cytokine signaling pathways. This profile overlapped with (chronic) dLck-Cre deletion model. Hence, we found that peripheral adaptation induced changes to both TCR and cytokine signaling modules. Our data highlight the importance of Themis in the activation of CD8+ T cells.
Asunto(s)
Linfocitos T CD8-positivos , Metabolismo Energético , Animales , Ratones , Citocinas , Receptores de Antígenos de Linfocitos T/genética , Tamoxifeno/farmacologíaRESUMEN
Serial passaging of the human fungal pathogen Candida albicans in the gastrointestinal tract of antibiotics-treated mice selects for virulence-attenuated strains. These gut-evolved strains protect the host from infection by a wide range of pathogens via trained immunity. Here, we further investigated the molecular and cellular mechanisms underlying this innate immune memory. Both Dectin-1 (the main receptor for ß-glucan; a well-described immune training molecule in the fungal cell wall) and Nod2 (a receptor described to mediate BCG-induced trained immunity), were redundant for the protection induced by gut-evolved C. albicans against a virulent C. albicans strain, suggesting that gut-evolved C. albicans strains induce trained immunity via other pathways. Cytometry by time of flight (CyTOF) analysis of mouse splenocytes revealed that immunization with gut-evolved C. albicans resulted in an expansion of neutrophils and a reduction in natural killer (NK) cells, but no significant numeric changes in monocytes, macrophages or dendritic cell populations. Systemic depletion of phagocytes or neutrophils, but not of macrophages or NK cells, reduced protection mediated by gut-evolved C. albicans. Splenocytes and bone marrow cells of mice immunized with gut-evolved C. albicans demonstrated metabolic changes. In particular, splenic neutrophils displayed significantly elevated glycolytic and respiratory activity in comparison to those from mock-immunized mice. Although further investigation is required for fully deciphering the trained immunity mechanism induced by gut-evolved C. albicans strains, this data is consistent with the existence of several mechanisms of trained immunity, triggered by different training stimuli and involving different immune molecules and cell types.
Asunto(s)
Candida albicans , beta-Glucanos , Animales , Pared Celular , Macrófagos , Ratones , NeutrófilosRESUMEN
The desmoplastic nature of the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) prevents the infiltration of T cells and the penetration of chemotherapeutic drugs, posing a challenge to the validation of targeted therapies, including T cell immunotherapies. We present an in vitro 3D PDAC-TME model to observe and quantify T cell infiltration across the vasculature. In a three-channel microfluidic device, PDAC cells are cultured in a collagen matrix in the central channel surrounded, on one side, by endothelial cells (ECs) to mimic a blood vessel and, on the opposite side, by pancreatic stellate cells (PSCs) to simulate exocrine pancreas. The migration of T cells toward the tumor is quantified based on their activation state and TME composition. The presence of EC-lining drastically reduces T cell infiltration, confirming the essential role of the vasculature in controlling T cell trafficking. We show that activated T cells migrate â¼50% more than the not-activated ones toward the cancer cells. Correspondingly, in the absence of cancer cells, both activated and not-activated T cells present similar migration toward the PSCs. The proposed approach could help researchers in testing and optimizing immunotherapies for pancreatic cancer.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Células Endoteliales , Humanos , Células Estrelladas Pancreáticas , Linfocitos T , Microambiente TumoralRESUMEN
The human fetal immune system begins to develop early during gestation; however, factors responsible for fetal immune-priming remain elusive. We explored potential exposure to microbial agents in utero and their contribution toward activation of memory T cells in fetal tissues. We profiled microbes across fetal organs using 16S rRNA gene sequencing and detected low but consistent microbial signal in fetal gut, skin, placenta, and lungs in the 2nd trimester of gestation. We identified several live bacterial strains including Staphylococcus and Lactobacillus in fetal tissues, which induced in vitro activation of memory T cells in fetal mesenteric lymph node, supporting the role of microbial exposure in fetal immune-priming. Finally, using SEM and RNA-ISH, we visualized discrete localization of bacteria-like structures and eubacterial-RNA within 14th weeks fetal gut lumen. These findings indicate selective presence of live microbes in fetal organs during the 2nd trimester of gestation and have broader implications toward the establishment of immune competency and priming before birth.
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Bacterias/metabolismo , Desarrollo Embrionario , Feto/citología , Feto/microbiología , Leucocitos/citología , Adulto , Bacterias/genética , Bacterias/ultraestructura , Proliferación Celular , Células Dendríticas/metabolismo , Femenino , Feto/ultraestructura , Tracto Gastrointestinal/embriología , Tracto Gastrointestinal/ultraestructura , Humanos , Memoria Inmunológica , Activación de Linfocitos/inmunología , Viabilidad Microbiana , Embarazo , Segundo Trimestre del Embarazo , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Reproducibilidad de los Resultados , Linfocitos T/citologíaRESUMEN
Macrophages are abundant in the tumor microenvironment (TME), serving as accomplices to cancer cells for their invasion. Studies have explored the biochemical mechanisms that drive pro-tumor macrophage functions; however the role of TME interstitial flow (IF) is often disregarded. Therefore, we developed a three-dimensional microfluidic-based model with tumor cells and macrophages to study how IF affects macrophage migration and its potential contribution to cancer invasion. The presence of either tumor cells or IF individually increased macrophage migration directedness and speed. Interestingly, there was no additive effect on macrophage migration directedness and speed under the simultaneous presence of tumor cells and IF. Further, we present an in silico model that couples chemokine-mediated signaling with mechanosensing networks to explain our in vitro observations. In our model design, we propose IL-8, CCL2, and ß-integrin as key pathways that commonly regulate various Rho GTPases. In agreement, in vitro macrophage migration remained elevated when exposed to a saturating concentration of recombinant IL-8 or CCL2 or to the co-addition of a sub-saturating concentration of both cytokines. Moreover, antibody blockade against IL-8 and/or CCL2 inhibited migration that could be restored by IF, indicating cytokine-independent mechanisms of migration induction. Importantly, we demonstrate the utility of an integrated in silico and 3D in vitro approach to aid the design of tumor-associated macrophage-based immunotherapeutic strategies.
Asunto(s)
Movimiento Celular , Quimiocinas/metabolismo , Inmunoterapia/métodos , Macrófagos/citología , Macrófagos/metabolismo , Microambiente Tumoral , Diferenciación Celular , Línea Celular Tumoral , Separación Celular , Técnicas de Cocultivo , Medios de Cultivo Condicionados , Citocinas/metabolismo , Humanos , Dispositivos Laboratorio en un Chip , Modelos Teóricos , Transducción de SeñalRESUMEN
Candida albicans is a ubiquitous fungal symbiont that resides on diverse human barrier surfaces. Both mammalian and fungal cells can convert arachidonic acid into the lipid mediator, prostaglandin E2 (PGE2), but the physiological significance of fungus-derived PGE2 remains elusive. Here we report that a C. albicans mutant deficient in PGE2 production suffered a loss of competitive fitness in the murine gastrointestinal (GI) tract and that PGE2 supplementation mitigated this fitness defect. Impaired fungal PGE2 production affected neither the in vitro fitness of C. albicans nor hyphal morphogenesis and virulence in either systemic or mucosal infection models. Instead, fungal production of PGE2 was associated with enhanced fungal survival within phagocytes. Consequently, ablation of colonic phagocytes abrogated the intra-GI fitness boost conferred by fungal PGE2. These observations suggest that C. albicans has evolved the capacity to produce PGE2 from arachidonic acid, a host-derived precursor, to promote its own colonization of the host gut. Analogous mechanisms might undergird host-microbe interactions of other symbiont fungi.
Asunto(s)
Dinoprostona/metabolismo , Hongos/fisiología , Interacciones Huésped-Patógeno , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Simbiosis , Animales , Candida albicans/fisiología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Mutación , Fagocitos/metabolismo , Virulencia/genéticaRESUMEN
Aneuploidy is common both in tumor cells responding to chemotherapeutic agents and in fungal cells adapting to antifungal drugs. Because aneuploidy simultaneously affects many genes, it has the potential to confer multiple phenotypes to the same cells. Here, we analyzed the mechanisms by which Candida albicans, the most prevalent human fungal pathogen, acquires the ability to survive both chemotherapeutic agents and antifungal drugs. Strikingly, adaptation to both types of drugs was accompanied by the acquisition of specific whole-chromosome aneuploidies, with some aneuploid karyotypes recovered independently and repeatedly from very different drug conditions. Specifically, strains selected for survival in hydroxyurea, an anticancer drug, acquired cross-adaptation to caspofungin, a first-line antifungal drug, and both acquired traits were attributable to trisomy of the same chromosome: loss of trisomy was accompanied by loss of adaptation to both drugs. Mechanistically, aneuploidy simultaneously altered the copy number of most genes on chromosome 2, yet survival in hydroxyurea or caspofungin required different genes and stress response pathways. Similarly, chromosome 5 monosomy conferred increased tolerance to both fluconazole and to caspofungin, antifungals with different mechanisms of action. Thus, the potential for cross-adaptation is not a feature of aneuploidy per se; rather, it is dependent on specific genes harbored on given aneuploid chromosomes. Furthermore, pre-exposure to hydroxyurea increased the frequency of appearance of caspofungin survivors, and hydroxyurea-adapted C. albicans cells were refractory to antifungal drug treatment in a mouse model of systemic candidiasis. This highlights the potential clinical consequences for the management of cancer chemotherapy patients at risk of fungal infections.
Asunto(s)
Aneuploidia , Antifúngicos , Antineoplásicos , Candida albicans/genética , Caspofungina , Farmacorresistencia Fúngica/genética , Hidroxiurea , Adaptación Biológica , Animales , Calcineurina , Cromosomas Fúngicos , RatonesRESUMEN
Gut microbes live in symbiosis with their hosts, but how mutualistic animal-microbe interactions emerge is not understood. By adaptively evolving the opportunistic fungal pathogen Candida albicans in the mouse gastrointestinal tract, we selected strains that not only had lost their main virulence program but also protected their new hosts against a variety of systemic infections. This protection was independent of adaptive immunity, arose as early as a single day postpriming, was dependent on increased innate cytokine responses, and was thus reminiscent of "trained immunity." Because both the microbe and its new host gain some advantages from their interaction, this experimental system might allow direct study of the evolutionary forces that govern the emergence of mutualism between a mammal and a fungus.
Asunto(s)
Inmunidad Adaptativa , Candida albicans/inmunología , Candida albicans/patogenicidad , Microbioma Gastrointestinal/inmunología , Tracto Gastrointestinal/microbiología , Interacciones Huésped-Patógeno , Animales , Evolución Biológica , Candida albicans/genética , Candida albicans/crecimiento & desarrollo , Proteínas Fúngicas/genética , Ratones , Ratones Endogámicos C57BL , Mutación , Simbiosis , Factores de Transcripción/genética , Factores de Virulencia/genéticaRESUMEN
Candida albicans is a resident fungus of the human intestinal microflora. Commonly isolated at low abundance in healthy people, C. albicans outcompetes local microbiota during candidiasis episodes. Under normal conditions, members of the human gastrointestinal (GI) microbiota were shown to keep C. albicans colonization under control. By releasing weak organic acids (WOAs), bacteria are able to moderate yeast growth. This mechanism displays a synergistic effect in vitro with the absence of glucose in medium of culture, which underlines the complex interactions that C. albicans faces in its natural environment. Inactivation of the transcriptional regulator MIG1 in C. albicans results in a lack of sensitivity to this synergistic outcome. To decipher C. albicans transcriptional responses to glucose, WOAs, and the role of MIG1, we performed RNA sequencing (RNA-seq) on four biological replicates exposed to combinations of these three parameters. We were able to characterize the (i) glucose response, (ii) response to acetic and butyric acid, (iii) MIG1 regulation of C. albicans, and (iv) genes responsible for WOA resistance. We identified a group of six genes linked to WOA sensitivity in a glucose-MIG1-dependent manner and inactivated one of these genes, the putative glucose transporter HGT16, in a SC5314 wild-type background. As expected, the mutant displayed a partial complementation to WOA resistance in the absence of glucose. This result points toward a mechanism of WOA sensitivity in C. albicans involving membrane transporters, which could be exploited to control yeast colonization in human body niches.
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Ácido Acético/farmacología , Antifúngicos/farmacología , Ácido Butírico/farmacología , Candida albicans/genética , Farmacorresistencia Fúngica/genética , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Glucosa/metabolismo , Ácido Acético/metabolismo , Antifúngicos/metabolismo , Ácido Butírico/metabolismo , Candida albicans/efectos de los fármacos , Candida albicans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , TranscriptomaRESUMEN
Candida albicans is responsible for ~400,000 systemic fungal infections annually, with an associated mortality rate of 46-75%. The human gastrointestinal (GI) tract represents the largest natural reservoir of Candida species and is a major source of systemic fungal infections. However, the factors that control GI colonization by Candida species are not completely understood. We hypothesized that the fungal cell wall would play an important role in determining the competitive fitness of Candida species in the mammalian GI tract. To test this hypothesis, we generated a systematic collection of isogenic C. albicans cell wall mutants and measured their fitness in the mouse GI tract via quantitative competition assays. Whereas a large variation in competitive fitness was found among mutants, no correlation was observed between GI fitness and total levels of individual cell wall components. Similar results were obtained in a set of distantly-related Candida species, suggesting that total amounts of individual cell wall components do not determine the ability of fungi to colonize the GI tract. We then subjected this collection of Candida strains and species to an extensive quantitative phenotypic profiling in search for features that might be responsible for their differences in GI fitness, but found no association with the ability to grow in GI-mimicking and stressful environments or with in vitro and in vivo virulence. The most significant association with GI fitness was found to be the strength of signaling through the Dectin-1 receptor. Using a quantitative assay to measure the amount of exposed ß-glucan on the surface of fungal cells, we found this parameter, unlike total ß-glucan levels, to be strongly predictive of competitive fitness in the mouse GI tract. These data suggest that fungal cell wall architecture, more so than its crude composition, critically determines the ability of fungi to colonize the mammalian GI tract. In particular, recognition of exposed ß-glucan by Dectin-1 receptor appears to severely limit Candida GI fitness and hence represents a promising target to reduce fungal colonization in patients at risks of systemic candidiasis.
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Candida albicans/química , Candida albicans/crecimiento & desarrollo , Pared Celular/química , Tracto Gastrointestinal/microbiología , beta-Glucanos/análisis , Animales , Lectinas Tipo C/metabolismo , RatonesRESUMEN
Candida albicans is the leading cause of fungal infections; but it is also a member of the human microbiome, an ecosystem of thousands of microbial species potentially influencing the outcome of host-fungal interactions. Accordingly, antibacterial therapy raises the risk of candidiasis, yet the underlying mechanism is currently not fully understood. We hypothesize the existence of bacterial metabolites that normally control C. albicans growth and of fungal resistance mechanisms against these metabolites. Among the most abundant microbiota-derived metabolites found on human mucosal surfaces are weak organic acids (WOAs), such as acetic, propionic, butyric, and lactic acid. Here, we used quantitative growth assays to investigate the dose-dependent fungistatic properties of WOAs on C. albicans growth and found inhibition of growth to occur at physiologically relevant concentrations and pH values. This effect was conserved across distantly related fungal species both inside and outside the CTG clade. We next screened a library of transcription factor mutants and identified several genes required for the resistance of C. albicans to one or more WOAs. A single gene, MIG1, previously known for its role in glucose repression, conferred resistance against all four acids tested. Consistent with glucose being an upstream activator of Mig1p, the presence of this carbon source was required for WOA resistance in wild-type C. albicans. Conversely, a MIG1-complemented strain completely restored the glucose-dependent resistance against WOAs. We conclude that Mig1p plays a central role in orchestrating a transcriptional program to fight against the fungistatic effect of this class of highly abundant metabolites produced by the gastrointestinal tract microbiota.
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Ácido Acético/farmacología , Antifúngicos/farmacología , Ácido Butírico/farmacología , Candida albicans/crecimiento & desarrollo , Proteínas Fúngicas/metabolismo , Ácido Láctico/farmacología , Propionatos/farmacología , Proteínas Represoras/metabolismo , Candida albicans/efectos de los fármacos , Candida albicans/genética , Candidiasis/microbiología , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Pruebas de Sensibilidad Microbiana , Proteínas Represoras/genéticaRESUMEN
Candida albicans is the most important fungal pathogen of humans, causing severe infections, especially in nosocomial and immunocompromised settings. However, it is also the most prevalent fungus of the normal human microbiome, where it shares its habitat with hundreds of trillions of other microbial cells. Despite weak organic acids (WOAs) being among the most abundant metabolites produced by bacterial microbiota, little is known about their effect on C. albicans. Here we used a sequencing-based profiling strategy to systematically investigate the transcriptional stress response of C. albicans to lactic, acetic, propionic, and butyric acid at several time points after treatment. Our data reveal a complex transcriptional response, with individual WOAs triggering unique gene expression profiles and with important differences between acute and chronic exposure. Despite these dissimilarities, we found significant overlaps between the gene expression changes induced by each WOA, which led us to uncover a core transcriptional response that was largely unrelated to other previously published C. albicans transcriptional stress responses. Genes commonly up-regulated by WOAs were enriched in several iron transporters, which was associated with an overall decrease in intracellular iron concentrations. Moreover, chronic exposure to any WOA lead to down-regulation of RNA synthesis and ribosome biogenesis genes, which resulted in significant reduction of total RNA levels and of ribosomal RNA in particular. In conclusion, this study suggests that gastrointestinal microbiota might directly influence C. albicans physiology via production of WOAs, with possible implications of how this fungus interacts with its host in both health and disease.