Tsc1 promotes the differentiation of memory CD8+ T cells via orchestrating the transcriptional and metabolic programs
ABSTRACT: Memory CD8+ T cells are an essential component of protective immunity. Signaling via mechanistic target of rapamycin (mTOR) has been implicated in the regulation of the differentiation of effector and memory T cells. However, little is understood about the mechanisms that control mTOR activity, or the effector pathways regulated by mTOR, in this process. We describe here that tuberous sclerosis 1 (Tsc1), a regulator of mTOR signaling, plays a crucial role in promoting the differentiation and function of memory CD8+ T cells in response to Listeria monocytogenes infection. Mice with specific deletion of Tsc1 in antigen-experienced CD8+ T cells evoked normal effector responses, but were markedly impaired in the generation of memory T cells and their recall responses to antigen re-exposure in a cell-intrinsic manner. Tsc1 deficiency suppressed the generation of memory-precursor effector cells (MPECs) while promoting short-lived effector cell (SLEC) differentiation. Functional genomic analysis indicated that Tsc1 coordinated gene expression programs underlying immune function, transcriptional regulation and cell metabolism. Furthermore, Tsc1 deletion led to excessive mTORC1 activity and dysregulated cellular metabolism including glycolytic and oxidative metabolism. These findings establish a Tsc1-mediated checkpoint in linking immune signaling and cell metabolism to orchestrate memory CD8+ T cell development and function. We used microarrays to explore the gene expression profiles differentially expressed in OVA-specific CD8+ T-cells from wild-type (WT; Tsc1-fl/fl and cre-negative) and Tsc1-/- (Tsc1-fl/fl and Granzyme B-cre-positive) 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:CD8 T cells play a crucial role in immunity to infection and cancer. They are maintained in constant numbers, but upon stimulation with antigen undergo a developmental program characterized by distinct phases encompassing the expansion and then contraction of antigen-specific populations, followed by the persistence of long-lived memory cells. Although this predictable pattern of a CD8 T cell response is well established, the underlying cellular mechanisms regulating the transition to memory remain undefined. Here we show that TRAF6, an adapter protein in the TNF-receptor (TNFR) and IL-1R/TLR superfamily, regulates CD8 T cell memory development following infection by modulating fatty acid metabolism. We show that mice with a T cell-specific deletion of TRAF6 mount robust primary CD8 T cell effector responses, but have a profound defect in their ability to generate memory. This defect is CD8 T cell intrinsic and is characterized by the disappearance of antigen-specific cells in the weeks following primary immunization. Microarray analyses revealed that TRAF6-deficient CD8 T cells from early timepoints following immunization exhibit altered expression of genes that regulate fatty acid metabolism. Consistent with this, activated CD8 T cells lacking TRAF6 are unable to upregulate mitochondrial β-oxidation in response to growth factor withdrawal in vitro. Treatment with drugs that induce fatty acid oxidation enabled CD8 T cell memory generation in the absence of TRAF6. Remarkably, these treatments also increased CD8 T cell memory in wild type mice, and consequently were able to significantly improve the efficacy of an experimental anti-cancer vaccine. Experiment Overall Design: CD8 T cells from two mouse strains (OTI-WT and OTI-TRAF6 knockout) at two timepoints (6d with 3 replicates and 10d with 5 replicates) after infection are used.
Project description:microRNA-150 (miR-150) is mainly expressed in the lymph nodes and spleen and is highly up-regulated during the development of mature T and B cells. To understand the signal-transduction network for 'effector or memory T cells' and 'mir-150' in naïve, effector, or memory CD8 T cells of mir-150 knockout and wild-type mice, we analyzed gene expression profiles by microarray. The genetic background of the cells used in this study is CD8 T cells from spleen of the 8 week-old male C57BL/6J mice (for female: WT-memory_2 & KO-memory_2). Corresponding authors: Inpyo Choi, PhD, Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro,Yuseong, Daejeon 305-806, Republic of Korea. E-mail: email@example.com. Or: Tae-Don Kim, PhD, Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong, Daejeon 305-806, Republic of Korea. E-mail: firstname.lastname@example.org. wild-type[WT] vs. mir-150 knockout[KO]; naïve, effector, and memory CD8 T cells.
Project description:ZEB2 is a multi-zinc-finger transcription factor known to play a significant role in early neurogenesis and in EMT-dependent tumor metastasis. While the function of ZEB2 in T lymphocytes is unknown, activity of the closely related family member ZEB1 has been implicated in lymphocyte development. Here, we find that ZEB2 expression is upregulated by activated T cells, specifically in the KLRG1hi effector CD8+ T cell subset. Loss of ZEB2 expression results in a significant loss of antigen-specific CD8+ T cells following primary and secondary infection with a severe impairment in the generation of the KLRG1hi effector-memory cell population. We show that ZEB2, which can bind DNA at tandem, consensus E-box sites, regulates gene expression of several E-protein targets and may directly repress CD127 and IL-2 in CD8+ T cells responding to infection. Furthermore, we find that T-bet binds to highly conserved T-box-sites in the ZEB2 gene and that T-bet and ZEB2 regulate similar gene-expression programs in effector T cells, suggesting that T-bet acts upstream and through regulation of ZEB2. Taken together, we place ZEB2 in a larger transcriptional network that is responsible for the balance between terminal differentiation and formation of memory CD8+ T cells. Zeb2WT, Zeb2KO, T-betWT and T-betKO effector CD8+ T cells were sorted into Trizol at day 6 of infection. Two or more replicates per sample were analyzed
Project description:Review on the role of Bcl11b in thymus and periphery and impact on immune diseases CD8+ T cells purified from bcl11bf/flckd-cre and wild type mice were infected with Listeria monocytogenes and on day 6 mice were sacrificed and Cd8+ T cells were purified at more than 90% purity. The RNA was purified with trizol and submitted for generation of library and microarrays to establish genes which are regulated in infected KO versus WT cytotoxic T cells.
Project description:Neonates often generate incomplete immunity against intracellular pathogens, although the mechanism of this defect is poorly understood. An important question is whether the impaired development of memory CD8+ T cells in neonates is due to an immature priming environment or lymphocyte-intrinsic defects. Here we show that neonatal and adult CD8+ T cells adopted different fates when responding to equal amounts of stimulation in the same host. While adult CD8+ T cells differentiated into a heterogeneous pool of effector and memory cells, neonatal CD8+ T cells preferentially gave rise to short-lived effector cells and exhibited a distinct gene expression profile. Surprisingly, impaired neonatal memory formation was not due to a lack of responsiveness, but instead because neonatal CD8+ T cells expanded more rapidly than adult cells and quickly became terminally differentiated. Collectively, these findings demonstrate that neonatal CD8+ T cells exhibit an imbalance in effector and memory CD8+ T cell differentiation, which impairs the formation of memory CD8+ T cells in early life mRNA profiles of effector CD8+ T cells from neonatal and adult mice
Project description:TCF-1 is an HMG family transcription factor which is known to be activated by the canonical Wnt signaling pathway and modulated by other signals such as those derived from T cell receptor. We found that during CD8 T cell responses, TCF-1 deficiency impaired long-term maintenance of antigen-specific memory CD8 T cells. We used microarrays to detect gene expression changes in memory CD8 T cells caused by TCF-1 deficiency. Host mice that received WT or TCF-1-deficient OT-I CD8 T cells were infected with attenuated Listeria monocytogenes expressing Ova peptide. In memory phase (i.e., more than 80 days post infection), antigen-specific T cells were ioslated by cell sorting. RNA was extracted and hybridized to GeneChip Mouse GENE 1.0 ST arrays (Affymetrix).
Project description:CD8+ T cells play a crucial role in the clearance of intracellular pathogens through the generation of cytotoxic effector cells that eliminate infected cells and long-lived memory cells that provide enhanced protection against reinfection. We have previously shown that the inhibitor of E protein transcription factors, Id2, is necessary for accumulation of effector and memory CD8+ T cells during infection. Here we show that CD8+ T cells lacking Id2 did not generate a robust terminally-differentiated KLRG1hi effector population, but displayed a cell-surface phenotype and cytokine profile consistent with memory precursors, raising the question as to whether loss of Id2 impairs the differentiation and/or survival of effector-memory cells. We found that deletion of Bim rescued Id2-deficient CD8+ cell survival during infection. However, the dramatic reduction in KLRG1hi cells caused by loss of Id2 remained in the absence of Bim, such that Id2/Bim double-deficient cells form an exclusively KLRG1loCD127hi memory precursor population. Thus we describe a role for Id2 in both the survival and differentation of normal CD8+ effector and memory populations. Gene-expression analysis of Wild-type, Id2KO, Id2KOBimKO and BimKO effector CD8+ cells on day 6 of Listeria infection. 2 or more replicates per sample were analyzed.
Project description:The precise timing and pathway of memory CD8+ T cells differentiation from naïve T cells have remained undetermined. We found the smaller cell-size and slower cell cycling cells were segregated from the proliferative larger cell-size activated T cell pool at the peak of infection. Gene signature of the smaller cell-size slower cycling cells and the large cell-size proliferative cells was compared to the signature of naïve, effector, central and effector memory CD8+ T cells. Total RNA samples were prepared from sorted populations of larger or smaller cell-sized cells from spleens of influenza virus PR8-OVA-infected mice on day 7 p.i. or from in vitro 7 days culture after stimulation with plate-bound anti-CD3ε (1.0 μg ml−1) and anti-CD28 mAb (0.5 μg ml−1). Effector T-cell control samples were prepared from SIINFEKL (100 ng ml−1) stimulated OT-I cells after 4 days of in vitro culture with rIL-2 (10 ng/ml) and sorted as CD8+CD44hiCD62Llo. Control bona fide effector memory and central memory T cells were sorted from the spleens of PR8-OVA-infected mice on day 42 p.i. Naive cells were sorted as CD8+CD44loCD62Lhi cells from uninfected C57BL/6 mice.
Project description:In this study we investigated the mechanisms involved in memory T-cell mediated protection using mice vaccinated with the intracellular bacterium Listeria monocytogenes. Our working hypothesis was that rapid activation of cells of the innate immune system, in particular inflammatory Ly6C+ monocytes, were essential in effective protection, in a memory T cell-dependent manner. Thus we generated a comprehensive comparison of the genetic program of activated Ly6C+ monocytes during a primary or a secondary infection with Listeria monocytogenes, at 8 hours post challenge infection. Abstract of corresponding publication: Cells of the innate immune system are essential for host defenses against primary microbial pathogen infections, yet their involvement in effective memory responses of vaccinated individuals has been poorly investigated. Here we show that memory T cells instruct innate cells to become potent effector cells in a systemic and a mucosal model of infection. Memory T cells controlled phagocyte, dendritic cell and NK or NK T cell mobilization and induction of a strong program of differentiation, which included their expression of effector cytokines and microbicidal pathways, all of which were delayed in non-vaccinated hosts. Disruption of IFN-gamma-signaling in Ly6C+ monocytes, dendritic cells and macrophages impaired these processes and the control of pathogen growth. These results reveal how memory T cells, through rapid secretion of IFN-gamma, orchestrate extensive modifications of host innate immune responses that are essential for effective protection of vaccinated hosts. Overall design: Inflammatory monocytes were purified (see below for isolation method) from 4 groups of 3 individual mice each (triplicate): (i) uninfected mice, (ii) primary infected, (iii) secondary infected, (iv) secondary infected and T-cell depleted 1 day before. Isolation of cells was done on 3 different days for true biological replicates.