RESUMEN
Memory B cells are central to the establishment of immunological memory, providing long-term protection against specific pathogens and playing a vital role in the efficacy of vaccines. Understanding how memory B cell formation is disrupted during persistent infection is essential for new therapeutics. Lymphocytic choriomeningitis virus (LCMV) is an ideal model for investigating memory B cells in acute versus chronic infection. This protocol details techniques to isolate, enrich, and examine LCMV-specific memory B cells in both acute and chronic LCMV infection. Using an antigen tetramer enrichment system and flow cytometry, this method assesses low-frequency, polyclonal antigen-specific memory B cells.
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Antígenos Virales , Citometría de Flujo , Coriomeningitis Linfocítica , Virus de la Coriomeningitis Linfocítica , Células B de Memoria , Virus de la Coriomeningitis Linfocítica/inmunología , Animales , Ratones , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Citometría de Flujo/métodos , Antígenos Virales/inmunología , Células B de Memoria/inmunología , Células B de Memoria/metabolismo , Memoria Inmunológica , Linfocitos B/inmunología , Linfocitos B/metabolismoAsunto(s)
Apirasa , Linfocitos T CD8-positivos , Linfocitos T CD8-positivos/inmunología , Animales , Ratones , Apirasa/metabolismo , Apirasa/inmunología , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Factor Nuclear 1-alfa del Hepatocito/genética , Humanos , Factor 1 de Transcripción de Linfocitos T/metabolismo , Factor 1 de Transcripción de Linfocitos T/genética , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virologíaRESUMEN
Immunotherapy with checkpoint inhibitors, albeit commonly used against tumors, is still at its infancy against chronic virus infections. It relies on the reinvigoration of exhausted T lymphocytes to eliminate virus-infected cells. Since T cell exhaustion is a physiological process to reduce immunopathology, the reinvigoration of these cells might be associated with an augmentation of pathological changes. To test this possibility, we here analyzed in the model system of chronic lymphocytic choriomeningitis virus (LCMV)-infected mice whether treatment with the checkpoint inhibitor anti-PD-L1 antibody would increase CD8 T cell-dependent fibrosis. We show that pre-existing spleen fibrosis did not worsen under conditions that increase CD8 T cell functionality and reduce virus loads suggesting that the CD8 T cell functionality increase remained below its pathogenicity threshold. These promising findings should further encourage immunotherapeutic trials against chronic virus infections.
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Antígeno B7-H1 , Linfocitos T CD8-positivos , Fibrosis , Inhibidores de Puntos de Control Inmunológico , Inmunoterapia , Coriomeningitis Linfocítica , Virus de la Coriomeningitis Linfocítica , Animales , Femenino , Ratones , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/inmunología , Enfermedad Crónica , Modelos Animales de Enfermedad , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , Coriomeningitis Linfocítica/complicaciones , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/terapia , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones Endogámicos C57BL , Bazo/inmunología , Bazo/virología , Carga ViralRESUMEN
Introduction: Macrophage function is determined by microenvironment and origin. Brain and retinal microglia are both derived from yolk sac progenitors, yet their microenvironments differ. Utilizing single-cell RNA sequencing (scRNA-seq) data from mice, we tested the hypothesis that retinal and brain microglia exhibit distinct transcriptional profiles due to their unique microenvironments. Methods: Eyes and brains from 2-4 month wildtype mice were combined (20 eyes; 3 brains) to yield one biologically diverse sample per organ. Each tissue was digested into single cell suspensions, enriched for immune cells, and sorted for scRNA-seq. Analysis was performed in Seurat v3 including clustering, integration, and differential expression. Multi-parameter flow cytometry was used for validation of scRNA-seq results. Lymphocytic choriomeningitis virus (LCMV) Clone 13, which produces a systemic, chronic, and neurotropic infection, was used to validate scRNA-seq and flow cytometry results in vivo. Results: Cluster analysis of integrated gene expression data from eye and brain identified 6 Tmem119 + P2ry12 + microglial clusters. Differential expression analysis revealed that eye microglia were enriched for more pro-inflammatory processes including antigen processing via MHC class I (14.0-fold, H2-D1 and H2-K1) and positive regulation of T-cell immunity (8.4-fold) compared to brain microglia. Multi-parameter flow cytometry confirmed that retinal microglia expressed 3.2-fold greater H2-Db and 263.3-fold more H2-Kb than brain microglia. On Day 13 and 29 after LCMV infection, CD8+ T-cell density was greater in the retina than the brain. Discussion: Our data demonstrate that the microenvironment of retina and brain differs, resulting in microglia-specific gene expression changes. Specifically, retinal microglia express greater MHC class I by scRNA-seq and multi-parameter flow cytometry, resulting in a possibly enhanced capability to stimulate CD8+ T-cell inflammation during LCMV infection. These results may explain tissue-specific differences between retina and brain during systemic viral infections and CD8+ T-cell driven autoimmune disease.
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Encéfalo , Microglía , Retina , Animales , Microglía/inmunología , Microglía/metabolismo , Ratones , Retina/inmunología , Retina/patología , Encéfalo/inmunología , Encéfalo/patología , Encéfalo/metabolismo , Ratones Endogámicos C57BL , Virus de la Coriomeningitis Linfocítica/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Linfocitos T/inmunología , Inflamación/inmunología , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Análisis de la Célula Individual , Linfocitos T CD8-positivos/inmunología , TranscriptomaRESUMEN
Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.
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Linfocitos T CD4-Positivos , Diferenciación Celular , Coriomeningitis Linfocítica , Animales , Ratones , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/inmunología , Citometría de Flujo/métodos , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones Endogámicos C57BL , Ratones Transgénicos , Células T Auxiliares Foliculares/inmunología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
The maintenance of antigen-specific CD8+ T cells underlies the efficacy of vaccines and immunotherapies. Pathways contributing to CD8+ T cell loss are not completely understood. Uncovering the pathways underlying the limited persistence of CD8+ T cells would be of significant benefit for developing novel strategies of promoting T cell persistence. Here, we demonstrate that murine CD8+ T cells experience endoplasmic reticulum (ER) stress following activation and that the ER-associated degradation (ERAD) adapter Sel1L is induced in activated CD8+ T cells. Sel1L loss limits CD8+ T cell function and memory formation following acute viral infection. Mechanistically, Sel1L is required for optimal bioenergetics and c-Myc expression. Finally, we demonstrate that human CD8+ T cells experience ER stress upon activation and that ER stress is negatively associated with improved T cell functionality in T cell-redirecting therapies. Together, these results demonstrate that ER stress and ERAD are important regulators of T cell function and persistence.
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Linfocitos T CD8-positivos , Estrés del Retículo Endoplásmico , Degradación Asociada con el Retículo Endoplásmico , Memoria Inmunológica , Animales , Humanos , Ratones , Enfermedad Aguda , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Coriomeningitis Linfocítica/patología , Ratones Endogámicos C57BL , Proteínas , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Masculino , FemeninoRESUMEN
Memory B cells (MBCs) are key providers of long-lived immunity against infectious disease, yet in chronic viral infection, they do not produce effective protection. How chronic viral infection disrupts MBC development and whether such changes are reversible remain unknown. Through single-cell (sc)ATAC-seq and scRNA-seq during acute versus chronic lymphocytic choriomeningitis viral infection, we identified a memory subset enriched for interferon (IFN)-stimulated genes (ISGs) during chronic infection that was distinct from the T-bet+ subset normally associated with chronic infection. Blockade of IFNAR-1 early in infection transformed the chromatin landscape of chronic MBCs, decreasing accessibility at ISG-inducing transcription factor binding motifs and inducing phenotypic changes in the dominating MBC subset, with a decrease in the ISG subset and an increase in CD11c+CD80+ cells. However, timing was critical, with MBCs resistant to intervention at 4 weeks post-infection. Together, our research identifies a key mechanism to instruct MBC identity during viral infection.
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Epigénesis Genética , Interferón Tipo I , Coriomeningitis Linfocítica , Virus de la Coriomeningitis Linfocítica , Células B de Memoria , Animales , Interferón Tipo I/metabolismo , Interferón Tipo I/inmunología , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Ratones , Virus de la Coriomeningitis Linfocítica/inmunología , Células B de Memoria/inmunología , Ratones Endogámicos C57BL , Receptor de Interferón alfa y beta/genética , Memoria Inmunológica/inmunología , Enfermedad Crónica , Subgrupos de Linfocitos B/inmunología , Análisis de la Célula IndividualRESUMEN
In response to acute infection, naive CD4+ T cells primarily differentiate into T helper 1 (Th1) or T follicular helper (Tfh) cells that play critical roles in orchestrating cellular or humoral arms of immunity, respectively. However, despite the well established role of T-bet and BCL-6 in driving Th1 and Tfh cell lineage commitment, respectively, whether additional transcriptional circuits also underlie the fate bifurcation of Th1 and Tfh cell subsets is not fully understood. In this article, we study how the transcriptional regulator Bhlhe40 dictates the Th1/Tfh differentiation axis in mice. CD4+ T cell-specific deletion of Bhlhe40 abrogates Th1 but augments Tfh differentiation. We also assessed an increase in germinal center B cells and Ab production, suggesting that deletion of Bhlhe40 in CD4+ T cells not only alters Tfh differentiation but also their capacity to provide help to B cells. To identify molecular mechanisms by which Bhlhe40 regulates Th1 versus Tfh lineage choice, we first performed epigenetic profiling in the virus specific Th1 and Tfh cells following LCMV infection, which revealed distinct promoter and enhancer activities between the two helper cell lineages. Furthermore, we identified that Bhlhe40 directly binds to cis-regulatory elements of Th1-related genes such as Tbx21 and Cxcr6 to activate their expression while simultaneously binding to regions of Tfh-related genes such as Bcl6 and Cxcr5 to repress their expression. Collectively, our data suggest that Bhlhe40 functions as a transcription activator to promote Th1 cell differentiation and a transcription repressor to suppress Tfh cell differentiation.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular , Células T Auxiliares Foliculares , Células TH1 , Animales , Ratones , Diferenciación Celular/inmunología , Diferenciación Celular/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células T Auxiliares Foliculares/inmunología , Células TH1/inmunología , Ratones Noqueados , Ratones Endogámicos C57BL , Linfocitos B/inmunología , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Centro Germinal/inmunología , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Virus de la Coriomeningitis Linfocítica/inmunología , Receptores CXCR5/genética , Receptores CXCR5/metabolismo , Proteínas de HomeodominioRESUMEN
Lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV) share many genetic and biological features including subtle differences between pathogenic and apathogenic strains. Despite remarkable genetic similarity, the viscerotropic WE strain of LCMV causes a fatal LASV fever-like hepatitis in non-human primates (NHPs) while the mouse-adapted Armstrong (ARM) strain of LCMV is deeply attenuated in NHPs and can vaccinate against LCMV-WE challenge. Here, we demonstrate that internalization of WE is more sensitive to the depletion of membrane cholesterol than ARM infection while ARM infection is more reliant on endosomal acidification. LCMV-ARM induces robust NF-κB and interferon response factor (IRF) activation while LCMV-WE seems to avoid early innate sensing and failed to induce strong NF-κB and IRF responses in dual-reporter monocyte and epithelial cells. Toll-like receptor 2 (TLR-2) signaling appears to play a critical role in NF-κB activation and the silencing of TLR-2 shuts down IL-6 production in ARM but not in WE-infected cells. Pathogenic LCMV-WE infection is poorly recognized in early endosomes and failed to induce TLR-2/Mal-dependent pro-inflammatory cytokines. Following infection, Interleukin-1 receptor-associated kinase 1 (IRAK-1) expression is diminished in LCMV-ARM- but not LCMV-WE-infected cells, which indicates it is likely involved in the LCMV-ARM NF-κB activation. By confocal microscopy, ARM and WE strains have similar intracellular trafficking although LCMV-ARM infection appears to coincide with greater co-localization of early endosome marker EEA1 with TLR-2. Both strains co-localize with Rab-7, a late endosome marker, but the interaction with LCMV-WE seems to be more prolonged. These findings suggest that LCMV-ARM's intracellular trafficking pathway may facilitate interaction with innate immune sensors, which promotes the induction of effective innate and adaptive immune responses.
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Inmunidad Innata , Virus de la Coriomeningitis Linfocítica , Internalización del Virus , Virus de la Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/patogenicidad , Virus de la Coriomeningitis Linfocítica/fisiología , Animales , Humanos , Ratones , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 2/genética , Endosomas/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Línea Celular , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Células Epiteliales/virología , Células Epiteliales/inmunologíaRESUMEN
Blindness or vision loss due to neuroretinal and photoreceptor degeneration affects millions of individuals worldwide. In numerous neurodegenerative diseases, including age-related macular degeneration, dysregulated immune response-mediated retinal degeneration has been found to play a critical role in the disease pathogenesis. To better understand the pathogenic mechanisms underlying the retinal degeneration, we used a mouse model of systemic immune activation where we infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13. Here, we evaluated the effects of LCMV infection and present a comprehensive discovery-based proteomic investigation using tandem mass tag (TMT) labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in protein regulation in the posterior part of the eye, neuroretina, and RPE/choroid were compared to those in the spleen as a secondary lymphoid organ and to the kidney as a non-lymphoid but encapsulated organ at 1, 8, and 28 weeks of infection. Using bioinformatic tools, we found several proteins responsible for maintaining normal tissue homeostasis to be differentially regulated in the neuroretina and the RPE/choroid during the degenerative process. Additionally, in the organs we observed, several important protein pathways contributing to cellular homeostasis and tissue development were perturbed and associated with LCMV-mediated inflammation, promoting disease progression. Our findings suggest that the response to a systemic chronic infection differs between the neuroretina and the RPE/choroid, and the processes induced by chronic systemic infection in the RPE/choroid are not unlike those induced in non-immune-privileged organs such as the kidney and spleen. Overall, our data provide detailed insight into several molecular mechanisms of neuroretinal degeneration and highlight various novel protein pathways that further suggest that the posterior part of the eye is not an isolated immunological entity despite the existence of neuroretinal immune privilege.
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Modelos Animales de Enfermedad , Virus de la Coriomeningitis Linfocítica , Proteómica , Degeneración Retiniana , Animales , Ratones , Proteómica/métodos , Degeneración Retiniana/inmunología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones Endogámicos C57BL , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Espectrometría de Masas en Tándem , Proteoma , Retina/inmunología , Retina/metabolismo , Retina/patología , Cromatografía Liquida , Coroides/inmunología , Coroides/patología , Coroides/metabolismoRESUMEN
Immune cells rely on transient physical interactions with other immune and non-immune populations to regulate their function1. To study these 'kiss-and-run' interactions directly in vivo, we previously developed LIPSTIC (labelling immune partnerships by SorTagging intercellular contacts)2, an approach that uses enzymatic transfer of a labelled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+ T helper cells and antigen-presenting cells, however. Here we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+ T cells by dendritic cells, reveal the steady-state cellular partners of regulatory T cells and identify germinal centre-resident T follicular helper cells on the basis of their ability to interact cognately with germinal centre B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalogue of the immune populations that physically interact with intestinal epithelial cells at the steady state and profile the evolution of the interactome of lymphocytic choriomeningitis virus-specific CD8+ T cells in multiple organs following systemic infection. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell-cell interactions across multiple biological systems.
Asunto(s)
Linfocitos B , Linfocitos T CD8-positivos , Comunicación Celular , Células Dendríticas , Células Epiteliales , Células T Auxiliares Foliculares , Linfocitos T Reguladores , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Comunicación Celular/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Ligandos , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Células T Auxiliares Foliculares/citología , Células T Auxiliares Foliculares/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Centro Germinal/citología , Análisis de Expresión Génica de una Sola Célula , Células Epiteliales/citología , Células Epiteliales/inmunología , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Especificidad de ÓrganosRESUMEN
T cell factor 1 (Tcf-1) expressing CD8+ T cells exhibit stem-like self-renewing capacity, rendering them key for immune defense against chronic viral infection and cancer. Yet, the signals that promote the formation and maintenance of these stem-like CD8+ T cells (CD8+SL) remain poorly defined. Studying CD8+ T cell differentiation in mice with chronic viral infection, we identified the alarmin interleukin-33 (IL-33) as pivotal for the expansion and stem-like functioning of CD8+SL as well as for virus control. IL-33 receptor (ST2)-deficient CD8+ T cells exhibited biased end differentiation and premature loss of Tcf-1. ST2-deficient CD8+SL responses were restored by blockade of type I interferon signaling, suggesting that IL-33 balances IFN-I effects to control CD8+SL formation in chronic infection. IL-33 signals broadly augmented chromatin accessibility in CD8+SL and determined these cells' re-expansion potential. Our study identifies the IL-33-ST2 axis as an important CD8+SL-promoting pathway in the context of chronic viral infection.
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Linfocitos T CD8-positivos , Interleucina-33 , Coriomeningitis Linfocítica , Animales , Ratones , Alarminas/metabolismo , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica , Ratones Endogámicos C57BL , Infección Persistente , Factor 1 de Transcripción de Linfocitos T/metabolismoRESUMEN
Increased demand for novel, highly effective vaccination strategies necessitates a better understanding of long-lived memory CD8 T cell differentiation. To achieve this understanding, we used the mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. We reexamined classical memory CD8 T cell subsets and performed in-depth, longitudinal analysis of their phenotype, transcriptional programming, and anatomic location within the spleen. All analyses were performed at multiple time points from 8 days to 1 year postinfection. Memory subsets are conventionally defined by their expression of KLRG1 and IL-7Rα, as follows: KLRG1+IL-7Rα- terminal effectors (TEs) and KLRG1-IL-7Rα+ memory precursors (MPs). But we also characterized a third KLRG1+IL-7Rα+ subset which we refer to as KLRG1+ MPs. In these analyses, we defined a comprehensive memory phenotype that is associated with higher levels of CD28 expression. We also demonstrated that MPs, KLRG1+ MPs, and TEs have distinct localization programs within the spleen. We found that MPs became preferentially enriched in the white pulp as early as 1 to 2 weeks postinfection, and their predominance in the white pulp was maintained throughout the course of a year. On the other hand, KLRG1+ MPs and TEs localized to the red pulp just as early, and they consistently localized to the red pulp thereafter. These findings indicate that location may be crucial for memory formation and that white pulp-derived signals may contribute to long-term memory survival. Achieving robust memory responses following vaccination may require more deliberate consideration of which memory phenotypes are induced, as well as where they traffic, as these factors could impact their longevity. IMPORTANCE CD8 T cells play a critical role in viral immunity and it is important to understand how memory cells are formed and what processes lead to their long-term maintenance. Here, we use a mouse model of acute infection to perform an in-depth, longitudinal analysis of memory CD8 T cell differentiation, examining the phenotype and location of memory cells out to 1 year postinfection.
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Coriomeningitis Linfocítica , Subgrupos de Linfocitos T , Animales , Ratones , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica , Ratones Endogámicos C57BL , Fenotipo , Vacunación , Antígenos CD28/genética , Transcriptoma , Antígenos de Superficie/genética , Vacunas Virales/inmunologíaRESUMEN
CD8 cytotoxic T cells are a potent line of defense against invading pathogens. To aid in curtailing aberrant immune responses, the activation status of CD8 T cells is highly regulated. One mechanism in which CD8 T cell responses are dampened is via signaling through the immune-inhibitory receptor Programmed Cell Death Protein-1, encoded by Pdcd1. Pdcd1 expression is regulated through engagement of the TCR, as well as by signaling from extracellular cytokines. Understanding such pathways has influenced the development of numerous clinical treatments. In this study, we showed that signals from the cytokine IL-6 enhanced Pdcd1 expression when paired with TCR stimulation in murine CD8 T cells. Mechanistically, signals from IL-6 were propagated through activation of the transcription factor STAT3, resulting in IL-6-dependent binding of STAT3 to Pdcd1 cis-regulatory elements. Intriguingly, IL-6 stimulation overcame B Lymphocyte Maturation Protein 1-mediated epigenetic repression of Pdcd1, which resulted in a transcriptionally permissive landscape marked by heightened histone acetylation. Furthermore, in vivo-activated CD8 T cells derived from lymphocytic choriomeningitis virus infection required STAT3 for optimal Programmed Cell Death Protein-1 surface expression. Importantly, STAT3 was the only member of the STAT family present at Pdcd1 regulatory elements in lymphocytic choriomeningitis virus Ag-specific CD8 T cells. Collectively, these data define mechanisms by which the IL-6/STAT3 signaling axis can enhance and prolong Pdcd1 expression in murine CD8 T cells.
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Linfocitos T CD8-positivos , Interleucina-6 , Receptor de Muerte Celular Programada 1 , Animales , Ratones , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Interleucina-6/metabolismo , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Virus de la Coriomeningitis Linfocítica/inmunologíaRESUMEN
Combination therapy with PD-1 blockade and IL-2 is highly effective during chronic lymphocytic choriomeningitis virus infection1. Here we examine the underlying basis for this synergy. We show that PD-1 + IL-2 combination therapy, in contrast to PD-1 monotherapy, substantially changes the differentiation program of the PD-1+TCF1+ stem-like CD8+ T cells and results in the generation of transcriptionally and epigenetically distinct effector CD8+ T cells that resemble highly functional effector CD8+ T cells seen after an acute viral infection. The generation of these qualitatively superior CD8+ T cells that mediate viral control underlies the synergy between PD-1 and IL-2. Our results show that the PD-1+TCF1+ stem-like CD8+ T cells, also referred to as precursors of exhausted CD8+ T cells, are not fate-locked into the exhaustion program and their differentiation trajectory can be changed by IL-2 signals. These virus-specific effector CD8+ T cells emerging from the stem-like CD8+ T cells after combination therapy expressed increased levels of the high-affinity IL-2 trimeric (CD25-CD122-CD132) receptor. This was not seen after PD-1 blockade alone. Finally, we show that CD25 engagement with IL-2 has an important role in the observed synergy between IL-2 cytokine and PD-1 blockade. Either blocking CD25 with an antibody or using a mutated version of IL-2 that does not bind to CD25 but still binds to CD122 and CD132 almost completely abrogated the synergistic effects observed after PD-1 + IL-2 combination therapy. There is considerable interest in PD-1 + IL-2 combination therapy for patients with cancer2,3, and our fundamental studies defining the underlying mechanisms of how IL-2 synergizes with PD-1 blockade should inform these human translational studies.
Asunto(s)
Linfocitos T CD8-positivos , Interleucina-2 , Receptor de Muerte Celular Programada 1 , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/efectos de los fármacos , Quimioterapia Combinada , Humanos , Subunidad gamma Común de Receptores de Interleucina , Interleucina-2/inmunología , Interleucina-2/farmacología , Interleucina-2/uso terapéutico , Subunidad alfa del Receptor de Interleucina-2 , Subunidad beta del Receptor de Interleucina-2 , Coriomeningitis Linfocítica/tratamiento farmacológico , Coriomeningitis Linfocítica/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Factor 1 de Transcripción de Linfocitos TRESUMEN
Chronic viral infections where the antigen persists long-term, induces an exhaustion phenotype in responding T cells. It is now evident that immune checkpoints on T cells including PD-1, CTLA-4, and PSGL-1 (Selplg) are linked with the differentiation of exhausted cells. Chronic T cell receptor signaling induces transcriptional signatures that result in the development of various exhausted T cell subsets, including the stem-like T cell precursor exhausted (Tpex) cells, which can be reinvigorated by immune checkpoint inhibitors (ICIs). While PSGL-1 has been shown to inhibit T cell responses in various disease models, the cell-intrinsic function of PSGL-1 in the differentiation, maintenance, and reinvigoration of exhausted T cells is unknown. We found Selplg-/- T cells had increased expansion in melanoma tumors and in early stages of chronic viral infection. Despite their increase, both WT and Selplg-/- T cells eventually became phenotypically and functionally exhausted. Even though virus-specific Selplg-/- CD4+ and CD8+ T cells were increased at the peak of T cell expansion, they decreased to lower levels than WT T cells at later stages of chronic infection. We found that Selplg-/- CD8+ Tpex (SLAMF6hiTIM3lo, PD-1+TIM3+, TOX+, TCF-1+) cell frequencies and numbers were decreased compared to WT T cells. Importantly, even though virus-specific Selplg-/- CD4+ and CD8+ T cells were lower, they were reinvigorated more effectively than WT T cells after anti-PD-L1 treatment. We found increased SELPLG expression in Hepatitis C-specific CD8+ T cells in patients with chronic infection, whereas these levels were decreased in patients that resolved the infection. Together, our findings showed multiple PSGL-1 regulatory functions in exhausted T cells. We found that PSGL-1 is a cell-intrinsic inhibitor that limits T cells in tumors and in persistently infected hosts. Additionally, while PSGL-1 is linked with T cell exhaustion, its expression was required for their long-term maintenance and optimal differentiation into Tpex cells. Finally, PSGL-1 restrained the reinvigoration potential of exhausted CD4+ and CD8+ T cells during ICI therapy. Our findings highlight that targeting PSGL-1 may have therapeutic potential alone or in combination with other ICIs to reinvigorate exhausted T cells in patients with chronic infections or cancer.
Asunto(s)
Linfocitos T CD8-positivos , Coriomeningitis Linfocítica , Glicoproteínas de Membrana , Receptor de Muerte Celular Programada 1 , Linfocitos T CD8-positivos/inmunología , Receptor 2 Celular del Virus de la Hepatitis A/inmunología , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Coriomeningitis Linfocítica/tratamiento farmacológico , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunologíaRESUMEN
Tissue-resident memory T cells (Trm) are retained in peripheral tissues after infection for enhanced protection against secondary encounter with the same pathogen. We have previously shown that the transcription factor Hobit and its homolog Blimp-1 drive Trm development after viral infection, but how and when these transcription factors mediate Trm formation remains poorly understood. In particular, the major impact of Blimp-1 in regulating several aspects of effector T-cell differentiation impairs study of its specific role in Trm development. Here, we used the restricted expression of Hobit in the Trm lineage to develop mice with a conditional deletion of Blimp-1 in Trm, allowing us to specifically investigate the role of both transcription factors in Trm differentiation. We found that Hobit and Blimp-1 were required for the upregulation of CD69 and suppression of CCR7 and S1PR1 on virus-specific Trm precursors after LCMV infection, underlining a role in their retention within tissues. The early impact of Hobit and Blimp-1 favored Trm formation and prevented the development of circulating memory T cells. Thus, our findings highlight a role of Hobit and Blimp-1 at the branching point of circulating and resident memory lineages by suppressing tissue egress of Trm precursors early during infection.
Asunto(s)
Linfocitos T CD8-positivos , Memoria Inmunológica , Coriomeningitis Linfocítica , Virus de la Coriomeningitis Linfocítica , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Factores de Transcripción , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/patología , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Factor 1 de Unión al Dominio 1 de Regulación Positiva/inmunología , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismoRESUMEN
Humoral immunity is a major component of the adaptive immune response against viruses and other pathogens with pathogen-specific antibody acting as the first line of defense against infection. Virus-specific antibody levels are maintained by continual secretion of antibody by plasma cells residing in the bone marrow. This raises the important question of how the virus-specific plasma cell population is stably maintained and whether memory B cells are required to replenish plasma cells, balancing their loss arising from their intrinsic death rate. In this study, we examined the longevity of virus-specific antibody responses in the serum of mice following acute viral infection with three different viruses: lymphocytic choriomeningitis virus (LCMV), influenza virus, and vesicular stomatitis virus (VSV). To investigate the contribution of memory B cells to the maintenance of virus-specific antibody levels, we employed human CD20 transgenic mice, which allow for the efficient depletion of B cells with rituximab, a human CD20-specific monoclonal antibody. Mice that had resolved an acute infection with LCMV, influenza virus, or VSV were treated with rituximab starting at 2 months after infection, and the treatment was continued for up to a year postinfection. This treatment regimen with rituximab resulted in efficient depletion of B cells (>95%), with virus-specific memory B cells being undetectable. There was an early transient drop in the antibody levels after rituximab treatment followed by a plateauing of the curve with virus-specific antibody levels remaining relatively stable (half-life of 372 days) for up to a year after infection in the absence of memory B cells. The number of virus-specific plasma cells in the bone marrow were consistent with the changes seen in serum antibody levels. Overall, our data show that virus-specific plasma cells in the bone marrow are intrinsically long-lived and can maintain serum antibody titers for extended periods of time without requiring significant replenishment from memory B cells. These results provide insight into plasma cell longevity and have implications for B cell depletion regimens in cancer and autoimmune patients in the context of vaccination in general and especially for COVID-19 vaccines. IMPORTANCE Following vaccination or primary virus infection, virus-specific antibodies provide the first line of defense against reinfection. Plasma cells residing in the bone marrow constitutively secrete antibodies, are long-lived, and can thus maintain serum antibody levels over extended periods of time in the absence of antigen. Our data, in the murine model system, show that virus-specific plasma cells are intrinsically long-lived but that some reseeding by memory B cells might occur. Our findings demonstrate that, due to the longevity of plasma cells, virus-specific antibody levels remain relatively stable in the absence of memory B cells and have implications for vaccination.
Asunto(s)
Anticuerpos Antivirales , Coriomeningitis Linfocítica , Células B de Memoria , Rituximab , Animales , Anticuerpos Antivirales/sangre , Humanos , Inmunidad Humoral , Memoria Inmunológica , Coriomeningitis Linfocítica/inmunología , Células B de Memoria/citología , Ratones , Ratones Transgénicos , Infecciones por Orthomyxoviridae/inmunología , Células Plasmáticas/citología , Infecciones por Rhabdoviridae/inmunología , Rituximab/farmacologíaRESUMEN
Type-I interferon (IFN-I) signals exert a critical role in disease progression during viral infections. However, the immunomodulatory mechanisms by which IFN-I dictates disease outcomes remain to be fully defined. Here we report that IFN-I signals mediate thymic atrophy in viral infections, with more severe and prolonged loss of thymic output and unique kinetics and subtypes of IFN-α/ß expression in chronic infection compared to acute infection. Loss of thymic output was linked to inhibition of early stages of thymopoiesis (DN1-DN2 transition, and DN3 proliferation) and pronounced apoptosis during the late DP stage. Notably, infection-associated thymic defects were largely abrogated upon ablation of IFNαßR and partially mitigated in the absence of CD8 T cells, thus implicating direct as well as indirect effects of IFN-I on thymocytes. These findings provide mechanistic underpinnings for immunotherapeutic strategies targeting IFN-1 signals to manipulate disease outcomes during chronic infections and cancers.
Asunto(s)
Atrofia/virología , Interferón-alfa/inmunología , Interferón beta/inmunología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Timocitos/virología , Timo/virología , Animales , Atrofia/genética , Atrofia/inmunología , Atrofia/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Enfermedad Crónica , Femenino , Regulación de la Expresión Génica , Humanos , Memoria Inmunológica , Interferón-alfa/genética , Interferón beta/genética , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Ganglios Linfáticos/virología , Depleción Linfocítica , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/patología , Virus de la Coriomeningitis Linfocítica/patogenicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/inmunología , Transducción de Señal/inmunología , Análisis de la Célula Individual , Timocitos/inmunología , Timocitos/patología , Timo/inmunología , Timo/patologíaRESUMEN
Recent studies have identified a critical role for B cell-produced cytokines in regulating both humoral and cellular immunity. Here, we show that B cells are an essential source of interleukin-27 (IL-27) during persistent lymphocytic choriomeningitis virus (LCMV) clone 13 (Cl-13) infection. By using conditional knockout mouse models with specific IL-27p28 deletion in B cells, we observed that B cell-derived IL-27 promotes survival of virus-specific CD4 T cells and supports functions of T follicular helper (Tfh) cells. Mechanistically, B cell-derived IL-27 promotes CD4 T cell function, antibody class switch, and the ability to control persistent LCMV infection. Deletion of IL-27ra in T cells demonstrated that T cell-intrinsic IL-27R signaling is essential for viral control, optimal CD4 T cell responses, and antibody class switch during persistent LCMV infection. Collectively, our findings identify a cellular mechanism whereby B cell-derived IL-27 drives antiviral immunity and antibody responses through IL-27 signaling on T cells to promote control of LCMV Cl-13 infection.