Therapeutic antiviral T cells noncytopathically clear persistently infected microglia after conversion into antigen-presenting cells
ABSTRACT: Several viruses can infect the mammalian nervous system and induce neurological dysfunction. Adoptive immunotherapy (AI) is an approach that involves administration of antiviral T cells and has shown promise in clinical studies for the treatment of peripheral virus infections in humans such as cytomegalovirus, Epstein-Barr virus, and adenovirus, among others. Clearance of neurotropic infections, on the other hand, is particularly challenging because the central nervous system (CNS) is relatively intolerant of immunopathological reactions. Therefore, it is essential to develop and mechanistically understand therapies that noncytopathically eradicate pathogens from the CNS. Here, we used mice persistently infected from birth with lymphocytic choriomeningitis virus (LCMV) to demonstrate that therapeutic antiviral T cells can completely purge the persistently infected brain without causing blood brain barrier breakdown or tissue damage. Mechanistically, this is accomplished through a tailored release of chemoattractants that recruit antiviral T cells, but few pathogenic innate immune cells such as neutrophils and inflammatory monocytes. Upon arrival, T cells enlisted the support of nearly all brain resident myeloid cells (microglia) by converting them into CD11c+ antigen-presenting cells (APCs) – a cell population also found in the brain of a human immunodeficiency virus infected patient. Two-photon imaging studies revealed that antiviral CD8+ and CD4+ T cells interacted directly with CD11c+ microglia and induced STAT1 signaling, but did not initiate programmed cell death. We propose that noncytopathic CNS viral clearance can be achieved by therapeutic antiviral T cells reliant on restricted chemoattractant production and interactions with apoptosis-resistant microglia. 6 Mouse Microglia-sorted Brain Samples: 3 (-) AI, 3 (+) AI.
Project description:CD4 T follicular helper (Tfh) cells provide the required signals to B cells for germinal center reactions that are necessary for longlived antibody responses. However, it remains unclear whether there are CD4+ memory T cells committed to the Tfh lineage after antigen clearance. Using adoptive transfer of antigen-specific memory CD4+ subpopulations (based on CXCR5 and Ly6c expression)in the LCMV infection model, we found that there are distinct memory CD4+ T cell populations with commitment to the Tfh and Th1 lineages. Our conclusions are based on gene expression profiles, epigenetic studies and phenotypic and functional analysis. The gene expression profiles of virus-specific CD4 T cell subets at effector and memory stages is presented here. The SMARTA TCR transgenic / adptive transfer system was used to identify and sort subsets of antigen-specific CD4 T cells (based on their expression of Ly6c and CXCR5) elicited after acute infection with LCMV (Arm).
Project description:Acquisition of effector properties is a key step in the generation of cytotoxic T lymphocytes (CTLs). Here we show that inflammatory signals regulate Dicer expression in CTL, and that deletion or depletion of Dicer in mouse or human activated CD8+ T cells causes upregulation of perforin, granzyme and effector cytokines. Genome-wide analysis of miRNA changes induced by exposure of differentiating CTLs to IL-2 and inflammatory signals identifies miR-139 and miR-150 as components of a miRNA network that controls perforin, eomesodermin (Eomes) and IL-2Ra expression in differentiating CTLs and whose activity is modulated by IL-2, inflammation and antigenic stimulation. Overall our data show that strong IL-2R and inflammatory signals act through Dicer and miRNAs to control the cytolytic program and other aspects of effector CTL differentiation. Comparison of control and Dicer knock-out CTLs differentiated in vitro; Comparison of wild type CTLs differentiated in vitro with or without inflammatory stimuli; Comparison of effector and memory precursor CTLs isolated from mice infected with LCMV-Armstrong
Project description:At the peak of the CD8 T cell response to acture viral and bacterial infections, expression of the Interleukin-7 Receptor (IL-7R) marks Memory Precursor Effector CD8 T Cells (MPECs) from other Short-Lived Effector CD8 T cells (SLECs), which are IL-7Rlo. This study was designed to determine the gene expression differences between these two subsets of effector CD8 T cells. Experiment Overall Design: This study compared IL-7Rhi and IL-7Rlo LCMV-specific P14 Transgenic CD8 T cells, sorted from LCMV armstrong infected recipient mice 6/7 days after infection. Data includes 3 independent replicates for the IL-7Rhi and IL-7Rlo groups.
Project description:In response to acute infection CD8 T cells differentiate into effector cells capable of clearing the antigen. While the transcriptional and functional changes have previously been studied little is known of the epigenetic modifications that accompany this differentiation process. To gain insights into CD8 T cell effector differentiation and the role of epigenetics, we mapped DNA methylation by MeDIP-seq in naive CD8 T cells and day 8 effector CD8 T cells that are induced following an acute infection. We identified hundreds of thousands of differentially methylated regions (DMRs). Promoter DNA methylation inversely correlated with gene expression and DMRs were enriched for functional transcription factor binding sites. These data indicated that DNA methylation is dynamic during CD8 T cell differentiation and provide a map of possible regulatory regions important in this process. Examination of DNA methylation during CD8 T cell differentiation from naïve to day 8 effectors following acute infection
Project description:During acute viral infections, effector CD8+ T cells differentiate into memory precursors or short-lived terminal effectors. miR-17-92a over-expression skews CD8+ effector cells to the terminal differentiation. We used microarray to identify the genes that are differentially expressed caused by miR-17-92a over-expression. CD8+ T cells from P14 TCR transgenic mice were infected with miR-17-92a-MSCV-IRES-Thy1.1 vector and transfer to C57BL6 recipients. Chimeras were infected with LCMV Armstrong. Thy1.1+ miR-17-92a-MSCV-IRES-Thy1.1 transduced P14 cells and Thy1.1- non-transduced P14 cells were sorted by FACS. RNA was extracted from samples, labeled, and hybridized to Affymetrix microarrays.
Project description:During acute viral infections, naïve CD8+ T cells differentiate into effector CD8+ T cells and, after viral control, into memory CD8+ T cells. Memory CD8+ T cells are highly functional, proliferate rapidly upon reinfection and persist long-term without antigen. In contrast, during chronic infections, CD8+ T cells become “exhausted” and have poor effector function, express multiple inhibitory receptors, possess low proliferative capacity, and cannot persist without antigen. To compare the development of functional memory T cells with poorly functional exhausted T cells, we generated longitudinal transcriptional profiles for each. Naive CD44Lo CD8+ T cells were isolated and sorted from uninfected C57BL/6 mice and H2-Db GP33-specific CD8+ T cells were sorted using MHC-I tetramers at d6, 8, 15, and 30 p.i. with either LCMV Arm or LCMV clone 13. RNA from these CD8+ T cells was processed, amplified, labeled, and hybridized to Affymetrix GeneChip MoGene 1.0 st microarrays
Project description:This SuperSeries is composed of the following subset Series: GSE34216: miRNA signatures of antigen specific CD8+ T cells at different stages of immune response to LCMV infection GSE34217: Expression profile of miR-17-92a-MSCV-IRES-Thy1.1 transduced P14 CD8+ T cells Refer to individual Series
Project description:The forkhead O transcription factors (FOXO) integrate a range of extracellular signals including growth factor signaling, inflammation, oxidative stress and nutrient availability, to substantially alter the program of gene expression and modulate cell survival, cell cycle progression, and many cell-type specific responses yet to be unraveled. Naive antigen-specific CD8+ T cells undergo a rapid expansion and arming of effector function within days of pathogen exposure, but in addition, by the peak of expansion, they form precursors to memory T cells capable of self-renewal and indefinite survival. We used microarrays to determine whether FOXO1 broadly affects effector and memory differentiation, and to what extent FOXO1 determines the program of memory T cell gene expression. To obtain an unbiased analysis of genes differentially expressed in antigen-specific Foxo1-/- CD8+ T cells responding to infection, we obtained RNA and performed Affymetrix microarray analysis from KLRG1low and KLRG1high FACS-sorted congenically-marked WT and Foxo1-/- P14 cells obtained from mixed transfers, eight days post-infection with LCMV-Armstrong. We carried out gene deletion in Rosa26Cre-ERT2 Foxo1f/f (Foxo1-/-) P14 mice just prior to adoptive transfer (Kerdiles et al., 2009), and transfer equal numbers of P14 cells from the spleens of KO (Foxo1-/- P14) and WT P14 mice. Day8 post infection
Project description:During acute viral infections, naïve CD4+ T cells differentiate into effector CD4+ T cells and, after viral control, into memory CD4+ T cells. Memory CD4+ T cells are highly functional, proliferate rapidly upon reinfection and persist long-term without antigen. In contrast, during chronic infections, CD4+ T cells become less functional. To compare the development of functional memory T cells with poorly functional T cells from chronic viral infection, we generated longitudinal transcriptional profiles for each. Naive CD44Lo CD4+ T cells were isolated and sorted from uninfected C57BL/6 mice and H2-IAb GP66-specific CD4+ T cells were sorted using MHC-II tetramers at d6, 8, 15, and 30 p.i. with either LCMV Arm or LCMV clone 13. RNA from these CD4+ T cells was processed, amplified, labeled, and hybridized to Affymetrix GeneChip MoGene 1.0 st microarrays.
Project description:Follicular helper T cells (TFH cells) are the prototypic helper T cell subset specialized to enable B cells to form germinal centers (GCs) and produce high-affinity antibodies. We found that expression of microRNAs (miRNAs) by T cells was essential for TFH cell differentiation. More specifically, we show that after immunization of mice with protein, the miRNA cluster miR-17~92 was critical for robust differentiation and function of TFH cells in a cell-intrinsic manner that occurred regardless of changes in proliferation. In a viral infection model, miR-17~92 restrained the expression of genes ‘inappropriate’ to the TFH cell subset, including the direct miR-17~92 target Rora. Removal of one Rora allele partially ‘rescued’ the inappropriate gene signature in miR-17~92-deficient TFH cells. Our results identify the miR-17~92 cluster as a critical regulator of T cell–dependent antibody responses, TFH cell differentiation and the fidelity of the TFH cell gene-expression program. Gene expression analysis of control versus miR-17~92 knockout (KO) LCMV-specific SMARTA TFH cells 5.5 days after viral infection.