Quiescence of Memory CD8+ T Cells Is Mediated by Regulatory T Cells through Inhibitory Receptor CTLA-4
ABSTRACT: Immune memory cells are poised to rapidly expand and elaborate effector functions upon reinfection. However, despite heightened readiness to respond, memory cells exist in a functionally quiescent state. The paradigm is that memory cells remain inactive due to lack of TCR stimuli. Here we report a unique role of Tregs in orchestrating memory quiescence by inhibiting effector and proliferation programs through CTLA-4. Loss of Tregs resulted in activation of genome-wide transcriptional programs characteristic of potent effectors, and both developing and established memory quickly reverted to a terminally differentiated (KLRG-1hi/IL-7R±lo/GzmBhi) phenotype, with compromised metabolic fitness, longevity, polyfunctionality and protective efficacy. CTLA-4, an inhibitory receptor overexpressed on Tregs, functionally replaced Tregs in trans to rescue Treg-less memory defects and restore homeostasis of secondary mediators as well. These studies present CD28-CTLA-4-CD80/CD86 axis as a novel target to potentially accelerate vaccine-induced immunity and improve T-cell memory quality in current cancer immunotherapies proposing transient Treg-depletion. We used microarray analysis to detail the global programming of gene expression in LCMV GP33-specific CD8 T cells differentiated in the presence or absence of regulatory T cells Differentiation of memory CD8 T cells entails a progressive transition of highly activated effector program to a quiescent memory program. A key question in the field is to understand the factors that aid in the differentiation of memory cells from effector cells. It is a generally accepted paradigm that effector cells transition to a memory state by default after antigen clearance, since TCR stimuli is the key driver of effector programs in CD8 T cells. We hypothesized that the effector to memory transition of CD8 T cells involves active immunological brakes through regulatory T cells (Tregs) that allow the highly activated effector cells to convert into quiescent memory cells. To address this hypothesis, we used FoxP3-DTR mice to deplete Tregs during the window following antigen clearance, during which the effector CD8 T cells convert to long-lived memory cells. To get a deeper understanding of the global transcriptome of CD8 T cells as they transition from an effector to a memory state, we isolated and arrayed the antigen-specific CD8 T cells at day 16 post-infection that have experienced the transitional environment with and without the presence of Tregs.
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: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. Exhuasted CD8+ T cells can be further segregated by their expression of the inhibitory cell surface receptor PD-1. We performed transcriptional profiling on both PD-1 High and PD-1 Intermediate H2-Db GP33-specific CD8+ T cells. H2-Db GP33-specific CD8+ T cells were sorted from C57BL/6 mice 30 days p.i. with LCMV clone 13. These cells were then segregated by their expression of the inhibitory cell surface receptor PD-1 into PD-1 High and PD-1 Intermediate subpopulations. We performed transcriptional profiling on these subpopulations.
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:Differentiation of CD8+ T lymphocytes into effector and memory cells is key for an adequate immune response and relies on complex interplay of pathways that convey signals from cell surface to nucleus. In this study, we fractionated four CD8+ T cell subtypes; naïve, recently activated effector, effector and memory cells into membrane, cytosol, soluble nucleus, chromatin-bound and cytoskeleton compartments. Using LC-MS/MS analysis, identified peptides were matched to human peptides/proteins (SwissProt). Compartment fractionation and gel-LC-MS separation identified 2399 proteins in total. Among these 735 were detected in all five, 241 in four, 257 in three, 368 in two and 798 found in only one fraction. Comparison between the two most different subsets, naïve and effector, yielded 146 significantly regulated proteins.
Project description:The maintenance of immune homeostasis requires regulatory T cells (Tregs). Given their intrinsic self-reactivity, Tregs must stably maintain a suppressive phenotype to avoid autoimmunity. We report that impaired expression of the transcription factor (TF) Helios by FoxP3+ CD4 and Qa-1-restricted CD8 Tregs results in defective regulatory activity and autoimmunity in mice. Helios-deficient Treg develop an unstable phenotype during inflammatory responses characterized by reduced FoxP3 expression and increased effector cytokine expression secondary to diminished activation of the STAT5 pathway. CD8 Treg also require Helios-dependent STAT5 activation for survival and to prevent terminal T cell differentiation. Definition of Helios as a key transcription factor that stabilizes regulatory T-cells in the face of inflammatory responses provides a genetic explanation for a core property of regulatory T-cells. We used microarrays to detail the global programs of gene expression by CD8 Treg (CD44+CD122+Ly49+) and conventional memory type of CD8 cells (CD44+CD122+Ly49-).
Project description:The ability to detect and isolate human CD8 TSP (Side population), Naïve, Effector memory (EM), Central memory (CM) cells allowed us to compare the global gene expression profiles of these cells. Human TSP cells comprise of distinct gene expression profile specifically enriched for genes overexpressed in TRM cells. RNA samples from CD8 TSP (Side population), Naïve, Effector memory (EM), Central memory (CM) cells were amplified, labeled, and hybridized on the Affymetrix Human Genome U133 Plus 2.0 microarray chips. The data were analyzed with GeneSpring GX 12.5 (Agilent Technologies)
Project description:DNMT3a is a de novo DNA methyltransferase expressed robustly after T cell activation that regulates plasticity of CD4+ T cell cytokine expression. Here we show that DNMT3a is critical for directing early CD8+ T cell effector and memory fate decisions. While effector function of DNMT3a knockout T cells is normal, they develop more memory precursor and fewer terminal effector cells in a T cell intrinsic manner compared to wild-type animals. Rather than increasing plasticity of differentiated effector CD8+ T cells, loss of DNMT3a biases differentiation of early effector cells into memory precursor cells. This is attributed in part to ineffective repression of Tcf1 expression in knockout T cells, as DNMT3a localizes to the Tcf7 promoter and catalyzes its de novo methylation in early effector WT CD8+ T cells. This data identifies DNMT3a as a crucial regulator of CD8+ early effector cell differentiation and effector versus memory fate decisions. Examination of global genomic DNA methylation by MBD-seq in naïve CD8 T cells and CD8 T cells 8 days post Vaccinia-Ova infection, comparing OT1 TCR-Tg CD8 T cells isolated from WT and T cell conditional DNMT3a KO mice.
Project description:T cells receive numerous positive and negative signals during primary antigen encounter that control their proliferation and function, but how these signals are integrated to modulate T cell memory has not been fully characterized. In these studies, we demonstrate that combining seemingly opposite signals, CTLA-4 blockade and rapamycin-mediated mTOR inhibition, during in vivo T cell priming leads to both an increase in the frequency of memory CD8+ T cells and improved memory responses to tumors and bacterial challenges. This enhanced efficacy corresponds to increased early expansion and memory precursor differentiation of CD8+ T cells and increased mitochondrial biogenesis and spare respiratory capacity in memory CD8+ T cells in mice treated with anti-CTLA-4 and rapamycin during immunization. Collectively, these results reveal that mTOR inhibition cooperates with rather than antagonizes blockade of CTLA-4, promoting unrestrained effector function and proliferation and an optimal metabolic program for CD8+ T cell memory. Total RNA was isolated from FACS-sorted, antigen-specific CD8+T cells from different treatment conditions at 5 or 35 days after primary T cell activation
Project description:During a T cell response, naïve CD8 T cells differentiate into effector cells. Subsequently, a subset of effector cells termed memory precursor effector cells (MPECs) further differentiates into functionally mature memory CD8 T cells. The transcriptional network underlying this carefully scripted process is not well understood. Here, we report that the transcription factor FoxO1 plays an integral role in facilitating effector to memory transition and functional maturation of memory CD4 and CD8 T cells. We find that FoxO1 is not required for differentiation of effector cells, but in the absence of FoxO1, memory CD8 T cells displayed features of scenescence and progressive attrition in polyfunctionality, which in turn led to impared recall responses and poor protective immunity. These data suggest that FoxO1 is essential for active maintenance of functional CD8 T cell memory and protective immunity. Under competing conditions in bone marrow Single-cell suspensions from splenocytes of eight samples WT (control) and FoxO1-/- (experimental) LCMV-immune mice were prepared using standard procedures. CD8 T cells were then isoloated using Thy1.2 (CD90.2) (30-H12) microbeads (Miltenyi Biotec). Cells were then stained with anti-CD8, anti-CD44 and Db/NP396 MHC class I tetramer. Activated (CD8+CD44hi), naive (CD8+CD44lo), and virus-specific CD8 T cells were sorted using FACSAria II instrument (BD Biosciences). The purity of the cells was >95%. Total RNA was extracted from the sorted cells by Trizol Reagent. RNA samples were reverse transcribed and Cy3-labeled cDNAs were hyrbidized to Agilent whole Mouse Genome Oligo Microarrays. Fluorscence signals were detected using Agilent's Microarray Scanner system, data was analyzed using the Rosetta Resolver gene expression data analysis system and genes with a fold change < and p-values <0.01 were identified. Microarray data discussed in the paper is focused on virus-specific memory CD8 T cells from samples WT_Tet_2 vs KO_Tet_2.
Project description:CD8 T cells normally differentiate from resting naïve T cells into function effector and then memory CD8 T cells following acute infections. During chronic viral infections, however, virus-specific CD8 T cells often become exhausted. We used microarrays to examine the gene expression differences between naive, effector, memory and exhausted virus-specific CD8 T cells following lymphocytic choriomeningitis virus infection. Experiment Overall Design: Three or four independent samples were sorted by flow cytometry for each cell type (naive, effector, memory and exhausted) virus-specific CD8 T cells. RNA was extracted and hybridized to Affymetrix microarrays.