MicroRNA-directed program of cytotoxic CD8+ T-cell differentiation.
ABSTRACT: 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 CTLs, and that deletion or depletion of Dicer in mouse or human activated CD8(+) T cells causes up-regulation of perforin, granzymes, and effector cytokines. Genome-wide analysis of microRNA (miR, miRNA) changes induced by exposure of differentiating CTLs to IL-2 and inflammatory signals identifies miR-139 and miR-150 as components of an miRNA network that controls perforin, eomesodermin, and IL-2R? 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.
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:Optimal T cell differentiation into effector cells with specialized functions requires the participation of cytokine receptor signals. In T helper cells, this process is controlled by chromatin changes and distal and proximal regulatory elements as well as specific transcription factors. Analogous events during cytotoxic T lymphocyte (CTL) differentiation remain to be identified. This process is known, however, to be crucially regulated by interleukin (IL)-2 receptor (R) signals. It is accompanied by the induction of perforin expression via a mechanism that does not entail proximal regulatory elements. In this report, transgenically expressed human perforin gene locus DNAs demonstrate that IL-2R signals target two IL-2-dependent enhancers approximately 15 and 1 kilobase upstream of the promoter. The most distal enhancer may also respond to TCR signals. In transient transfections, both enhancers required two identically spaced Stat-like elements for their activation, which was abolished by expression of a dominant negative signal transducer and activator of transcription (Stat)5 molecule, whereas a constitutively active Stat5 molecule bypassed the requirement for IL-2R signals. These results provide a molecular explanation for the activation of the perforin gene during CTL differentiation and complement the analysis of animals deficient in the activation of the IL-2R Stat signaling pathway by establishing perforin as a target gene.
Project description:Activation of naive CD8(+) T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs). CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes. We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) gamma expression, respectively. In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation. Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-gamma, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.
Project description:Cytolytic CD4 T cells (CD4 CTL) have been identified in vivo in response to viral infections; however, the factors necessary for driving the cytolytic phenotype have not been fully elucidated. Our previously published work suggests IL-2 may be the master regulator of perforin-mediated cytotoxicity in CD4 effectors. To further dissect the role of IL-2 in CD4 CTL generation, T cell receptor transgenic mice deficient in the ability to produce IL-2 or the high affinity IL-2 receptor (IL-2R?, CD25) were used. Increasing concentrations of IL-2 were necessary to drive perforin (Prf) expression and maximal cytotoxicity. Granzyme B (GrB) expression and killing correlated with STAT5 activation and CD25 expression in vitro, suggesting that signaling through the high affinity IL-2R is critical for full cytotoxicity. IL-2 signaling was also necessary in vivo for inducing the Th1 phenotype and IFN-? expression in CD4 T cells during influenza A (IAV) infection. In addition, GrB expression, as measured by mean fluorescent intensity, was decreased in CD25 deficient cells; however, the frequency of CD4 cells expressing GrB was unchanged. Similarly, analysis of cytolytic markers such as CD107a/b and Eomesodermin indicate high IL-2R? expression is not necessary to drive the CD4 CTL phenotype during IAV infection. Thus, inflammatory signals induced by viral infection may overcome the need for strong IL-2 signals in driving cytotoxicity in CD4 cells.
Project description:Interleukin(IL)-2 and inflammation regulate effector and memory cytolytic T-lymphocyte (CTL) generation during infection. We demonstrate a complex interplay between IL-2 and inflammatory signals during CTL differentiation. IL-2 stimulation induced the transcription factor eomesodermin (Eomes), upregulated perforin (Prf1) transcription, and repressed re-expression of memory CTL markers Bcl6 and IL-7Ralpha. Binding of Eomes and STAT5 to Prf1 cis-regulatory regions correlated with transcriptional initiation (increased recruitment of RNA polymerase II to the Prf1 promoter). Inflammation (CpG, IL-12) enhanced expression of IL-2Ralpha and the transcription factor T-bet, but countered late Eomes and perforin induction while preventing IL-7Ralpha repression by IL-2. After infection of mice with lymphocytic choriomeningitis virus, IL-2Ralpha-deficient effector CD8(+) T cells expressed more Bcl6 but less perforin and granzyme B, formed fewer KLRG-1(+) and T-bet-expressing CTL, and killed poorly. Thus, inflammation influences both effector and memory CTL differentiation, whereas persistent IL-2 stimulation promotes effector at the expense of memory CTL development.
Project description:Interleukin-2 (IL-2) is a pleiotropic cytokine that regulates immune cell homeostasis and has been used to treat a range of disorders including cancer and autoimmune disease. IL-2 signals via interleukin-2 receptor-? (IL-2R?):IL-2R? heterodimers on cells expressing high (regulatory T cells, Treg) or low (effector cells) amounts of IL-2R? (CD25). When complexed with IL-2, certain anti-cytokine antibodies preferentially stimulate expansion of Treg (JES6-1) or effector (S4B6) cells, offering a strategy for targeted disease therapy. We found that JES6-1 sterically blocked the IL-2:IL-2R? and IL-2:IL-2R? interactions, but also allosterically lowered the IL-2:IL-2R? affinity through a "triggered exchange" mechanism favoring IL-2R?(hi) Treg cells, creating a positive feedback loop for IL-2R?(hi) cell activation. Conversely, S4B6 sterically blocked the IL-2:IL-2R? interaction, while also conformationally stabilizing the IL-2:IL-2R? interaction, thus stimulating all IL-2-responsive immune cells, particularly IL-2R?(hi) effector cells. These insights provide a molecular blueprint for engineering selectively potentiating therapeutic antibodies.
Project description:Human CD8(+) effector T cells derived from CD45RO(+)CD62L(+) precursors enriched for central memory (TCM) precursors retain the capacity to engraft and reconstitute functional memory upon adoptive transfer, whereas effectors derived from CD45RO(+)CD62L(-) precursors enriched for effector memory precursors do not. Here we sought to compare the engraftment fitness and function of CD8(+) effector T cells derived from CD45RA(+)CD62L(+) precursors enriched for naïve and stem cell memory precursors (TN/SCM) with that of TCM. We found that cytotoxic T cells (CTLs) derived from TCM transcribed higher levels of CD28, FOS, INF?, Eomesodermin (Eomes), and lower levels of BCL2L11, maintained higher levels of phosphorylated AKT, and displayed enhanced sensitivity to the proliferative and anti-apoptotic effects of ?-chain cytokines compared to CTLs derived from TN/SCM. Higher frequencies of CTLs derived from TCM retained CD28 expression and upon activation secreted higher levels of IL-2. In NOD/Scid IL-2R?C(null) mice, CD8(+) TCM derived CTLs engrafted to higher frequencies in response to human IL-15 and mounted robust proliferative responses to an immunostimulatory vaccine. Similarly, CD8(+) TCM derived CD19CAR(+) CTLs exhibited superior antitumor potency following adoptive transfer compared to their CD8(+) TN/SCM derived counterparts. These studies support the use of TCM enriched cell products for adoptive therapy of cancer.
Project description:The maturation of naive CD8(+) T cells into effector CTLs is a critical feature of a functional adaptive immune system. Development of CTLs depends, in part, upon the expression of the transcriptional regulator eomesodermin (EOMES), which is thought to regulate expression of two key effector molecules, perforin and granzyme B. Although EOMES is important for effector CTL development, the precise mechanisms regulating CD8(+) effector cell maturation remains poorly understood. In this study, we show that Notch1 regulates the expression of EOMES, perforin, and granzyme B through direct binding to the promoters of these crucial effector molecules. By abrogating Notch signaling, both biochemically as well as genetically, we conclude that Notch activity mediates CTL activity through direct regulation of EOMES, perforin, and granzyme B.
Project description:Much is known concerning the cellular and molecular basis for CD8(+) T memory immune responses. Nevertheless, conditions that selectively support memory generation have remained elusive. In this study, we show that an immunization regimen that delivers TCR signals through a defined antigenic peptide, inflammatory signals through LPS, and growth and differentiation signals through the IL-2R initially favors Ag-specific CD8(+) T cells to develop rapidly and substantially into T effector-memory cells by TCR transgenic OVA-specific OT-I CD8(+) T cells. Amplified CD8(+) T memory development depends upon a critical frequency of Ag-specific T cells and direct responsiveness to IL-2. A homologous prime-boost immunization protocol with transiently enhanced IL-2R signaling in normal mice led to persistent polyclonal Ag-specific CD8(+) T cells that supported protective immunity to Listeria monocytogenes. These results identify a general approach for amplified T memory development that may be useful to optimize vaccines aimed at generating robust cell-mediated immunity.
Project description:CD4+ T cell help is required for the generation of CD8+ cytotoxic T lymphocyte (CTL) memory. Here, we use genome-wide analyses to show how CD4+ T cell help delivered during priming promotes memory differentiation of CTLs. Help signals enhance IL-15-dependent maintenance of central memory T (TCM) cells. More importantly, help signals regulate the size and function of the effector memory T (TEM) cell pool. Helped TEM cells produce Granzyme B and IFN? upon antigen-independent, innate-like recall by IL-12 and IL-18. In addition, helped memory CTLs express the effector program characteristic of helped primary CTLs upon recall with MHC class I-restricted antigens, likely due to epigenetic imprinting and sustained mRNA expression of effector genes. Our data thus indicate that during priming, CD4+ T cell help optimizes CTL memory by creating TEM cells with innate and help-independent antigen-specific recall capacities.