RESUMEN
Mammalian arenaviruses are rodent-borne zoonotic viruses, some of which can cause fatal hemorrhagic diseases in humans. The first discovered arenavirus, lymphocytic choriomeningitis virus (LCMV), has a worldwide distribution and can be fatal for transplant recipients. However, no FDA-approved drugs or vaccines are currently available. In this study, using a quantitative proteomic analysis, we identified a variety of host factors that could be needed for LCMV infection, among which we found that protein disulfide isomerase A4 (PDIA4), a downstream factor of endoplasmic reticulum stress (ERS), is important for LCMV infection. Biochemical analysis revealed that LCMV glycoprotein was the main viral component accounting for PDIA4 upregulation. The inhibition of ATF6-mediated ERS could prevent the upregulation of PDIA4 that was stimulated by LCMV infection. We further found that PDIA4 can affect the LCMV viral RNA synthesis processes and release. In summary, we conclude that PDIA4 could be a new target for antiviral drugs against LCMV.
Asunto(s)
Coriomeningitis Linfocítica , Virus de la Coriomeningitis Linfocítica , Animales , Humanos , Glicoproteínas , Coriomeningitis Linfocítica/metabolismo , Mamíferos , Proteína Disulfuro Isomerasas , ProteómicaRESUMEN
Acute and chronic viral infections result in the differentiation of effector and exhausted T cells with functional and phenotypic differences that dictate whether the infection is cleared or progresses to chronicity. High CD38 expression has been observed on CD8+ T cells across various viral infections and tumors in patients, suggesting an important regulatory function for CD38 on responding T cells. Here, we show that CD38 expression was increased and sustained on exhausted CD8+ T cells following chronic lymphocytic choriomeningitis virus (LCMV) infection, with lower levels observed on T cells from acute LCMV infection. We uncovered a cell-intrinsic role for CD38 expression in regulating the survival of effector and exhausted CD8+ T cells. We observed increased proliferation and function of Cd38-/- CD8+ progenitor exhausted T cells compared to those of wild-type (WT) cells. Furthermore, decreased oxidative phosphorylation and glycolytic potential were observed in Cd38-/- CD8+ T cells during chronic but not acute LCMV infection. Our studies reveal that CD38 has a dual cell-intrinsic function in CD8+ T cells, where it decreases proliferation and function yet supports their survival and metabolism. These findings show that CD38 is not only a marker of T cell activation but also has regulatory functions on effector and exhausted CD8+ T cells. IMPORTANCE Our study shows how CD38 expression is regulated on CD8+ T cells responding during acute and chronic viral infection. We observed higher CD38 levels on CD8+ T cells during chronic viral infection compared to levels during acute viral infection. Deleting CD38 had an important cell-intrinsic function in ensuring the survival of virus-specific CD8+ T cells throughout the course of viral infection. We found defective metabolism in Cd38-/- CD8+ T cells arising during chronic infection and changes in their progenitor T cell phenotype. Our studies revealed a dual cell-intrinsic role for CD38 in limiting proliferation and granzyme B production in virus-specific exhausted T cells while also promoting their survival. These data highlight new avenues for research into the mechanisms through which CD38 regulates the survival and metabolism of CD8+ T cell responses to viral infections.
Asunto(s)
Coriomeningitis Linfocítica , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Diferenciación Celular/genética , Coriomeningitis Linfocítica/metabolismo , Virus de la Coriomeningitis Linfocítica/genética , Infección Persistente , Animales , Ratones , Supervivencia Celular/genética , Regulación hacia Arriba , Proliferación Celular/genéticaRESUMEN
T cell responses are regulated by co-stimulatory and inhibitory receptors along with T cell receptor- and cytokine-mediated signals. CD51 is a transmembrane glycoprotein of the integrin family that plays a role in cell adhesion, migration, tumorigenesis, and other cellular functions. In this study, we aimed to investigate the expression and function of CD51 on CD8 T cells. Upon in vitro T cell activation, CD51 expression was delayed but subsequently was upregulated in CD8 T cells upon cell division. Furthermore, CD51 was highly expressed in exhausted CD8 T cells in chronic LCMV infection, B16F10 melanoma, and CT26 colon carcinoma, and its expression level increased as cells became more differentiated. Using CRISPR-mediated knockdown, we found that the absence of CD51 led to a lower number of virus-specific CD8 T cells upon chronic lymphocytic choriomeningitis virus (LCMV) infection, although their granzyme B expression and cytokine production were maintained. Blocking CD51 also inhibited the in vitro proliferation of CD8 T cells. These results suggest that CD51 plays an important role in the early expansion of CD8 T cells and may have potential as an immunomodulatory target.
Asunto(s)
Coriomeningitis Linfocítica , Animales , Ratones , Linfocitos T CD8-positivos , Citocinas/metabolismo , Activación de Linfocitos , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/patología , Virus de la Coriomeningitis Linfocítica , Ratones Endogámicos C57BL , Integrina alfaV/inmunologíaRESUMEN
Diacylglycerol is a potent element of intracellular secondary signaling cascades whose production is enhanced by cell-surface receptor agonism and function is regulated by enzymatic degradation by diacylglycerol kinases (DGKs). In T cells, stringent regulation of the activity of this second messenger maintains an appropriate balance between effector function and anergy. In this article, we demonstrate that DGKα is an indispensable regulator of TCR-mediated activation of CD8 T cells in lymphocytic choriomeningitis virus Clone 13 viral infection. In the absence of DGKα, Clone 13 infection in a murine model results in a pathologic, proinflammatory state and a multicellular immunopathologic host death that is predominantly driven by CD8 effector T cells.
Asunto(s)
Coriomeningitis Linfocítica , Virus de la Coriomeningitis Linfocítica , Ratones , Animales , Diacilglicerol Quinasa/genética , Diacilglicerol Quinasa/metabolismo , Diglicéridos/metabolismo , Coriomeningitis Linfocítica/metabolismo , Linfocitos T CD8-positivos/metabolismo , Células Clonales , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Reinvigorating the function of exhausted CD8+ T cells during chronic viral infection and cancer is a major goal of current immunotherapy regimens. Here, we discuss recent advances in our understanding of exhausted CD8+ T cell heterogeneity as well as the potential differentiation trajectories that exhausted T cells follow during chronic infection and/or cancer. We highlight surmounting evidence suggesting that some T cell clones are divergent in nature and can develop into either terminally differentiated effector or exhausted CD8+ T cells. Lastly, we consider the potential therapeutic implications of such a bifurcation model of CD8+ T cell differentiation, including the intriguing hypothesis that redirecting progenitor CD8+ T cell differentiation along an effector pathway may serve as a novel approach to mitigate T cell exhaustion.
Asunto(s)
Coriomeningitis Linfocítica , Neoplasias , Humanos , Linfocitos T CD8-positivos/metabolismo , Coriomeningitis Linfocítica/metabolismo , Virus de la Coriomeningitis Linfocítica/fisiología , Diferenciación CelularRESUMEN
During a microbial infection, responding CD8+ T cells give rise to effector cells that provide acute host defense and memory cells that provide sustained protection. An alternative outcome is exhaustion, a state of T cell dysfunction that occurs in the context of chronic infections and cancer. Although it is evident that exhausted CD8+ T (TEX) cells are phenotypically and molecularly distinct from effector and memory CD8+ T cells, the factors regulating the earliest events in the differentiation process of TEX cells remain incompletely understood. Here, we performed single-cell RNA-sequencing and single-cell ATAC-sequencing of CD8+ T cells responding to LCMV-Armstrong (LCMV-Arm) or LCMV-Clone 13 (LCMV-Cl13), which result in acute or chronic infections, respectively. Compared to CD8+ T cells that had undergone their first division in response to LCMV-Arm (Div1ARM) cells, CD8+ T cells that had undergone their first division in response to LCMV-Cl13 (Div1CL13) expressed higher levels of genes encoding transcription factors previously associated with exhaustion, along with higher levels of Ezh2, the catalytic component of the Polycomb Repressive Complex 2 (PRC2) complex, which mediates epigenetic silencing. Modulation of Ezh2 resulted in altered expression of exhaustion-associated molecules by CD8+ T cells responding to LCMV-Cl13, though the specific cellular and infectious contexts, rather than simply the level of Ezh2 expression, likely determine the eventual outcome. Taken together, these findings suggest that the differentiation paths of CD8+ T cells responding to acute versus chronic infections may diverge earlier than previously appreciated.
Asunto(s)
Coriomeningitis Linfocítica , Humanos , Animales , Ratones , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/metabolismo , Infección Persistente , Linfocitos T CD8-positivos/metabolismo , Virus de la Coriomeningitis Linfocítica , Epigénesis Genética , Ratones Endogámicos C57BLRESUMEN
Naïve CD8+ T cells can differentiate into effector (Teff), memory (Tmem) or exhausted (Tex) T cells. These developmental pathways are associated with distinct transcriptional and epigenetic changes that endow cells with different functional capacities and therefore therapeutic potential. The molecular circuitry underlying these developmental trajectories and the extent of heterogeneity within Teff, Tmem and Tex populations remain poorly understood. Here, we used the lymphocytic choriomeningitis virus model of acute-resolving and chronic infection to address these gaps by applying longitudinal single-cell RNA-sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses. These analyses uncovered new subsets, including a subpopulation of Tex cells expressing natural killer cell-associated genes that is dependent on the transcription factor Zeb2, as well as multiple distinct TCF-1+ stem/progenitor-like subsets in acute and chronic infection. These data also revealed insights into the reshaping of Tex subsets following programmed death 1 (PD-1) pathway blockade and identified a key role for the cell stress regulator, Btg1, in establishing the Tex population. Finally, these results highlighted how the same biological circuits such as cytotoxicity or stem/progenitor pathways can be used by CD8+ T cell subsets with highly divergent underlying chromatin landscapes generated during different infections.
Asunto(s)
Linfocitos T CD8-positivos , Coriomeningitis Linfocítica , Humanos , Linfocitos T CD8-positivos/metabolismo , Transcriptoma , Virus de la Coriomeningitis Linfocítica , Epigénesis Genética , Cromatina/genética , Cromatina/metabolismo , Coriomeningitis Linfocítica/metabolismoRESUMEN
Bleeding correlates with disease severity in viral hemorrhagic fevers. We found that the increase in type I interferon (IFN-I) in mice caused by infection with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV; an arenavirus) reduced the megakaryocytic expression of genes encoding enzymes involved in lipid biosynthesis (cyclooxygenase 1 and thromboxane A synthase 1) and a thrombopoietic transcription factor (Nf-e2). The decreased expression of these genes was associated with reduced numbers of circulating platelets and defects in the arachidonic acid synthetic pathway, thereby suppressing serotonin release from δ-granules in platelets. Bleeding resulted when severe thrombocytopenia and altered platelet function reduced the amount of platelet-derived serotonin below a critical threshold. Bleeding was facilitated by the absence of the activity of the kinase Lyn or the administration of aspirin, an inhibitor of arachidonic acid synthesis. Mouse platelets were not directly affected by IFN-I because they lack the receptor for the cytokine (IFNAR1), suggesting that transfusion of normal platelets into LCMV-infected mice could increase the amount of platelet-released serotonin and help to control hemorrhage.
Asunto(s)
Coriomeningitis Linfocítica , Animales , Plaquetas/metabolismo , Hemorragia/metabolismo , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/metabolismo , Virus de la Coriomeningitis Linfocítica/genética , Ratones , Serotonina/metabolismoRESUMEN
CD4+ T cells have a remarkable potential to differentiate into diverse effector lineages following activation. Here, we probe the heterogeneity present among naive CD4+ T cells before encountering their cognate antigen to ask whether their effector potential is modulated by pre-existing transcriptional and chromatin landscape differences. Single-cell RNA sequencing shows that key drivers of variability are genes involved in T cell receptor (TCR) signaling. Using CD5 expression as a readout of the strength of tonic TCR interactions with self-peptide MHC, and sorting on the ends of this self-reactivity spectrum, we find that pre-existing transcriptional differences among naive CD4+ T cells impact follicular helper T (TFH) cell versus non-TFH effector lineage choice. Moreover, our data implicate TCR signal strength during thymic development in establishing differences in naive CD4+ T cell chromatin landscapes that ultimately shape their effector potential.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular , Cromatina/fisiología , Activación de Linfocitos/inmunología , Coriomeningitis Linfocítica/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Femenino , Perfilación de la Expresión Génica , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Masculino , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Inhibiting PD-1:PD-L1 signaling has transformed therapeutic immune restoration. CD4+ T cells sustain immunity in chronic infections and cancer, yet little is known about how PD-1 signaling modulates CD4+ helper T (TH) cell responses or the ability to restore CD4+ TH-mediated immunity by checkpoint blockade. We demonstrate that PD-1:PD-L1 specifically suppressed CD4+ TH1 cell amplification, prevents CD4+ TH1 cytokine production and abolishes CD4+ cytotoxic killing capacity during chronic infection in mice. Inhibiting PD-L1 rapidly restored these functions, while simultaneously amplifying and activating TH1-like T regulatory cells, demonstrating a system-wide CD4-TH1 recalibration. This effect coincided with decreased T cell antigen receptor signaling, and re-directed type I interferon (IFN) signaling networks towards dominant IFN-γ-mediated responses. Mechanistically, PD-L1 blockade specifically targeted defined populations with pre-established, but actively suppressed proliferative potential, with limited impact on minimally cycling TCF-1+ follicular helper T cells, despite high PD-1 expression. Thus, CD4+ T cells require unique differentiation and functional states to be targets of PD-L1-directed suppression and therapeutic restoration.
Asunto(s)
Antígeno B7-H1/antagonistas & inhibidores , Inhibidores de Puntos de Control Inmunológico/farmacología , Activación de Linfocitos/efectos de los fármacos , Coriomeningitis Linfocítica/tratamiento farmacológico , Virus de la Coriomeningitis Linfocítica/inmunología , Células TH1/efectos de los fármacos , Traslado Adoptivo , Animales , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Proliferación Celular/efectos de los fármacos , Enfermedad Crónica , Citocinas/metabolismo , Citotoxicidad Inmunológica/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Redes Reguladoras de Genes , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/patogenicidad , Ratones Endogámicos C57BL , Fenotipo , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Células TH1/inmunología , Células TH1/metabolismo , Células TH1/virología , TranscriptomaRESUMEN
Chronic viral infections are associated with hematopoietic suppression, bone marrow (BM) failure, and hematopoietic stem cell (HSC) exhaustion. However, how persistent viral challenge and inflammatory responses target BM tissues and perturb hematopoietic competence remains poorly understood. Here, we combine functional analyses with advanced 3D microscopy to demonstrate that chronic infection with lymphocytic choriomeningitis virus leads to (1) long-lasting decimation of the BM stromal network of mesenchymal CXCL12-abundant reticular cells, (2) proinflammatory transcriptional remodeling of remaining components of this key niche subset, and (3) durable functional defects and decreased competitive fitness in HSCs. Mechanistically, BM immunopathology is elicited by virus-specific, activated CD8 T cells, which accumulate in the BM via interferon-dependent mechanisms. Combined antibody-mediated inhibition of type I and II IFN pathways completely preempts degeneration of CARc and protects HSCs from chronic dysfunction. Hence, viral infections and ensuing immune reactions durably impact BM homeostasis by persistently decreasing the competitive fitness of HSCs and disrupting essential stromal-derived, hematopoietic-supporting cues.
Asunto(s)
Médula Ósea/virología , Células Madre Hematopoyéticas/patología , Células Madre Hematopoyéticas/virología , Coriomeningitis Linfocítica/patología , Animales , Médula Ósea/metabolismo , Médula Ósea/patología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/virología , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Enfermedad Crónica , Regulación de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Interferones/metabolismo , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/virología , Ratones Endogámicos C57BL , Ratones Mutantes , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/inmunología , Receptor de Interferón alfa y beta/metabolismoRESUMEN
Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s).
Asunto(s)
Proteoglicanos de Heparán Sulfato/metabolismo , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/transmisión , Virus de la Coriomeningitis Linfocítica/metabolismo , Virus de la Coriomeningitis Linfocítica/patogenicidad , Células HEK293 , Humanos , Receptores Virales/metabolismoRESUMEN
CD8+ T cells are major components of adaptive immunity and confer robust protective cellular immunity, which requires adequate T-cell numbers, targeted migration, and efficient T-cell proliferation. Altered CD8+ T-cell homeostasis and impaired proliferation result in dysfunctional immune response to infection or tumorigenesis. However, intrinsic factors controlling CD8+ T-cell homeostasis and immunity remain largely elusive. Here, we demonstrate the prominent role of Brd4 on CD8+ T cell homeostasis and immune response. By upregulating Myc and GLUT1 expression, Brd4 facilitates glucose uptake and energy production in mitochondria, subsequently supporting naïve CD8+ T-cell survival. Besides, Brd4 promotes the trafficking of naïve CD8+ T cells partially through maintaining the expression of homing receptors (CD62L and LFA-1). Furthermore, Brd4 is required for CD8+ T cell response to antigen stimulation, as Brd4 deficiency leads to a severe defect in clonal expansion and terminal differentiation by decreasing glycolysis. Importantly, as JQ1, a pan-BRD inhibitor, severely dampens CD8+ T-cell immune response, its usage as an anti-tumor agent or latency-reversing agent for human immunodeficiency virus type I (HIV-1) should be more cautious. Collectively, our study identifies a previously-unexpected role of Brd4 in the metabolic regulation of CD8+ T cell-mediated immune surveillance and also provides a potential immunomodulation target.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD8-positivos/virología , Proliferación Celular , Activación de Linfocitos , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Traslado Adoptivo , Animales , Azepinas/farmacología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/trasplante , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Modelos Animales de Enfermedad , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Glucólisis , Interacciones Huésped-Patógeno , Activación de Linfocitos/efectos de los fármacos , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/metabolismo , Virus de la Coriomeningitis Linfocítica/patogenicidad , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/inmunología , Mitocondrias/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Triazoles/farmacología , Células VeroRESUMEN
Mast cells (MCs), strategically localized at mucosal surfaces, provide first-line defense against pathogens and shape innate and adaptive immune responses. Recent studies have shown that MCs are involved in pathogenic responses to several viruses including herpes simplex viruses, dengue virus, vaccinia virus and influenza virus. However, the underlying mechanisms of MCs in the activation of CD8+ T cells during viral infections are not fully understood. Therefore, we investigate the role of MCs in the development of virus-specific CD8+ T cell responses using the well-characterized murine lymphocytic choriomeningitis virus (LCMV) model and the transgenic MasTRECK mice that contain the human diphtheria toxin receptor as an inducible MC-deficient model. Here, we report that MCs are essential for the activation and expansion of virus-specific CD8+ T cells. After MC depletion and subsequent intradermal LCMV infection, the CD8 + T cell effector phenotype and antiviral cytokine production were impaired at the peak of infection (day 8 p.i.). Importantly, MC-deficient mice were unable to control the infection and exhibited significantly higher viral loads in the spleen and in the ear draining lymph nodes compared to that of wild type control mice. In the absence of MCs, dendritic cell (DC) activation was impaired upon LCMV infection. In addition, type-I interferon (IFN) levels in the serum and in the spleen of MC-deficient mice were reduced during the first days of infection. Interestingly, depletion of MCs after intradermal LCMV infection did not impair virus-specific CD8+ T cell expansion, activation or antiviral cytokine production. In summary, our results indicate that MCs play a pivotal role in the activation and antiviral functions of CD8+ T cells through proper DC activation. A better understanding of the impact of MCs on CD8+ T cell responses is mandatory to improve antiviral immune responses.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Activación de Linfocitos , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Mastocitos/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Comunicación Celular , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/virología , Modelos Animales de Enfermedad , Factor de Crecimiento Similar a EGF de Unión a Heparina/genética , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Interacciones Huésped-Patógeno , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/patogenicidad , Mastocitos/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Transducción de SeñalRESUMEN
The BTB zinc finger transcription factor MAZR (also known as PATZ1) controls, partially in synergy with the transcription factor Runx3, the development of CD8 lineage T cells. Here we explored the role of MAZR as well as combined activities of MAZR/Runx3 during cytotoxic T lymphocyte (CTL) and memory CD8+ T cell differentiation. In contrast to the essential role of Runx3 for CTL effector function, the deletion of MAZR had a mild effect on the generation of CTLs in vitro. However, a transcriptome analysis demonstrated that the combined deletion of MAZR and Runx3 resulted in much more widespread downregulation of CTL signature genes compared to single Runx3 deletion, indicating that MAZR partially compensates for loss of Runx3 in CTLs. Moreover, in line with the findings made in vitro, the analysis of CTL responses to LCMV infection revealed that MAZR and Runx3 cooperatively regulate the expression of CD8α, Granzyme B and perforin in vivo. Interestingly, while memory T cell differentiation is severely impaired in Runx3-deficient mice, the deletion of MAZR leads to an enlargement of the long-lived memory subset and also partially restored the differentiation defect caused by loss of Runx3. This indicates distinct functions of MAZR and Runx3 in the generation of memory T cell subsets, which is in contrast to their cooperative roles in CTLs. Together, our study demonstrates complex interplay between MAZR and Runx3 during CTL and memory T cell differentiation, and provides further insight into the molecular mechanisms underlying the establishment of CTL and memory T cell pools.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Subunidad alfa 3 del Factor de Unión al Sitio Principal/inmunología , Memoria Inmunológica/inmunología , Proteínas de Neoplasias/inmunología , Proteínas Represoras/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Regulación de la Expresión Génica/inmunología , Interacciones Huésped-Patógeno/inmunología , Activación de Linfocitos/inmunología , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones Noqueados , Ratones Transgénicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/virologíaRESUMEN
Regulatory T cells (Tregs) prevent excessive immune responses and limit immune pathology upon infections. To fulfill this role in different immune environments elicited by different types of pathogens, Tregs undergo functional specialization into distinct subsets. During acute type 1 immune responses, type 1 Tregs are induced and recruited to the site of ongoing Th1 responses to efficiently control Th1 responses. However, whether a similar specialization process also takes place following chronic infections is still unknown. In this study, we investigated Treg specialization in persistent viral infections using lymphocytic choriomeningitis virus (LCMV) and murine cytomegalovirus (MCMV) infection as models for chronic and latent infections, respectively. We identify CD85k as a Th1-specific co-inhibitory receptor with sustained expression in persistent viral infections and show that recombinant CD85k inhibits LCMV-specific effector T cells. Furthermore, expression of the CD85k ligand ALCAM is induced on LCMV-specific and exhausted T cells during chronic LCMV infection. Finally, we demonstrate that type 1 Tregs arising during chronic LCMV infection suppress Th1 effector cells in an ALCAM-dependent manner. These results extend the current knowledge of Treg specialization from acute to persistent viral infections and reveal an important functional role of CD85k in Treg-mediated suppression of type 1 immunity.
Asunto(s)
Virus de la Coriomeningitis Linfocítica/inmunología , Glicoproteínas de Membrana/inmunología , Muromegalovirus/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígenos CD/inmunología , Antígenos CD/metabolismo , Moléculas de Adhesión Celular Neuronal/inmunología , Moléculas de Adhesión Celular Neuronal/metabolismo , Línea Celular , Células Cultivadas , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/virología , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/fisiología , Glicoproteínas de Membrana/metabolismo , Ratones Endogámicos C57BL , Muromegalovirus/fisiología , Receptores Inmunológicos/metabolismo , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/virología , Células TH1/inmunología , Células TH1/metabolismoRESUMEN
The role of Ly49+CD8 T-cells in the immune system is not clear. Previously, several papers suggested Ly49+CD8 T-cells as immunosuppressors, while multiple studies also suggested their role as potent participants of the immune response. The mechanism of Ly49 expression on CD8 T-cells is also not clear. We investigated phenotype, functions, and regulation of Ly49 expression on murine CD8 T-cells in both normal state and during LCMV infection. CD8 T-cells express different Ly49 receptors compared with NK-cells. In intact mice, Ly49+CD8 T-cells have a phenotype similar to resting central memory CD8 T-cells and do not show impaired proliferation and cytokine production. Conventional CD8 T-cells upregulate Ly49 receptors during TCR-induced stimulation, and IL-2, as well as IL-15, affect it. At the same time, Ly49+CD8 T-cells change the Ly49 expression profile dramatically upon re-stimulation downregulating inhibitory and upregulating activating Ly49 receptors. We observed the expression of Ly49 receptors on the virus-specific CD8 T-cells during LCMV infection, especially marked in the early stages, and participation of Ly49+CD8 T-cells in the anti-viral response. Thus, CD8 T-cells acquire Ly49 receptors during the T-cell activation and show dynamic regulation of Ly49 receptors during stimulation.
Asunto(s)
Linfocitos T CD8-positivos/virología , Activación de Linfocitos , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/patogenicidad , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Traslado Adoptivo , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/trasplante , Proliferación Celular , Chlorocebus aethiops , Citocinas/metabolismo , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno , Memoria Inmunológica , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/metabolismo , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Subfamilia A de Receptores Similares a Lectina de Células NK/genética , Fenotipo , Transducción de Señal , Células VeroRESUMEN
During prolonged exposure to Ags, such as chronic viral infections, sustained TCR signaling can result in T cell exhaustion mediated in part by expression of programmed cell death-1 (PD-1) encoded by the Pdcd1 gene. In this study, dynamic changes in histone H3K4 modifications at the Pdcd1 locus during ex vivo and in vivo activation of CD8 T cells suggested a potential role for the histone H3 lysine 4 demethylase LSD1 in regulating PD-1 expression. CD8 T cells lacking LSD1 expressed higher levels of Pdcd1 mRNA following ex vivo stimulation as well as increased surface levels of PD-1 during acute, but not chronic, infection with lymphocytic choriomeningitis virus (LCMV). Blimp-1, a known repressor of PD-1, recruited LSD1 to the Pdcd1 gene during acute, but not chronic, LCMV infection. Loss of DNA methylation at Pdcd1's promoter-proximal regulatory regions is highly correlated with its expression. However, following acute LCMV infection, in which PD-1 expression levels return to near baseline, LSD1-deficient CD8 T cells failed to remethylate the Pdcd1 locus to the levels of wild-type cells. Finally, in a murine melanoma model, the frequency of PD-1-expressing tumor-infiltrating LSD1-deficient CD8 T cells was greater than in wild type. Thus, LSD1 is recruited to the Pdcd1 locus by Blimp-1, downregulates PD-1 expression by facilitating the removal of activating histone marks, and is important for remethylation of the locus. Together, these data provide insight into the complex regulatory mechanisms governing T cell immunity and regulation of a critical T cell checkpoint gene.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Histona Demetilasas/metabolismo , Coriomeningitis Linfocítica/metabolismo , Virus de la Coriomeningitis Linfocítica/fisiología , Melanoma/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Acetilación , Enfermedad Aguda , Animales , Enfermedad Crónica , Regulación de la Expresión Génica , Histona Demetilasas/genética , Histonas/metabolismo , Activación de Linfocitos/genética , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Experimentales , Receptor de Muerte Celular Programada 1/genética , Transducción de SeñalRESUMEN
Viral late domains are used by many viruses to recruit the cellular endosomal sorting complex required for transport (ESCRT) to mediate membrane scission during viral budding. Unlike the P(S/T)AP and YPX(1-3)L late domains, which interact directly with the ESCRT proteins Tsg101 and ALIX, the molecular linkage connecting the PPXY late domain to ESCRT proteins is unclear. The mammarenavirus lymphocytic choriomeningitis virus (LCMV) matrix protein, Z, contains only one late domain, PPXY. We previously found that this domain in LCMV Z, as well as the ESCRT pathway, are required for the release of defective interfering (DI) particles but not infectious virus. To better understand the molecular mechanism of ESCRT recruitment by the PPXY late domain, affinity purification-mass spectrometry was used to identify host proteins that interact with the Z proteins of the Old World mammarenaviruses LCMV and Lassa virus. Several Nedd4 family E3 ubiquitin ligases interact with these matrix proteins and in the case of LCMV Z, the interaction was PPXY-dependent. We demonstrated that these ligases directly ubiquitinate LCMV Z and mapped the specific lysine residues modified. A recombinant LCMV containing a Z that cannot be ubiquitinated maintained its ability to produce both infectious virus and DI particles, suggesting that direct ubiquitination of LCMV Z alone is insufficient for recruiting ESCRT proteins to mediate virus release. However, Nedd4 ligases appear to be important for DI particle release suggesting that ubiquitination of targets other than the Z protein itself is required for efficient viral ESCRT recruitment.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/fisiología , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Ubiquitinación , Ensamble de Virus , Replicación Viral , Humanos , Coriomeningitis Linfocítica/metabolismo , Dominios Proteicos , Dominios y Motivos de Interacción de ProteínasRESUMEN
Generation of protective immunity to infections and vaccinations declines with age. Studies in healthy individuals have implicated reduced miR-181a expression in T cells as contributing to this defect. To understand the impact of miR-181a expression on antiviral responses, we examined LCMV infection in mice with miR-181ab1-deficient T cells. We found that miR-181a deficiency delays viral clearance, thereby biasing the immune response in favor of CD4 over CD8 T cells. Antigen-specific CD4 T cells in mice with miR-181a-deficient T cells expand more and have a broader TCR repertoire with preferential expansion of high-affinity T cells than in wild-type mice. Importantly, generation of antigen-specific miR-181a-deficient CD8 effector T cells is particularly impaired, resulting in lower frequencies of CD8 T cells in the liver even at time points when the infection has been cleared. Consistent with the mouse model, CD4 memory T cells in individuals infected with West Nile virus at older ages tend to be more frequent and of higher affinity.