t cell activation and differentation to memory t cell

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ritika.saxena on Jul 13, 2012 Says :

Elucidative information about blood cells

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Advanced immunology course Develpoment of effectory and memory cells after Antigenic stimulation By: Amin Reza Nikpoor Ph.D candidate (90) 91.04.05 ?? ??? ?????? ?????? ? ?????? 1

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TCR complex & Ag recognition Costimulators of T cell activation Signaling pathways of T cell activation Changes in T cell after activation T cell differentiation Developing Memory cells Hemostasis of T cells 2

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Recognition of Ag: Antigen: first signal for the activation of lymphocytes, ensuring that the resultant immune response remains specific for the antigen. Activation of naive T cells requires recognition of antigen presented by dendritic cells. The initial activation of naive T lymphocytes :mainly in secondary lymphoid organs. from several hours to a few days, antigen-bearing, CCR7-expressing DCs will migrate from infected tissues through the lymphatic vessels in a CCL21-dependent process . 3

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Dendritic cells that have encountered microbes and internalized their antigens begin to mature and migrate to the T cell zones of draining lymph nodes. 4

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Secondary lymphoid tissues : focal point of an adaptive immune response because they unite anticipatory naïve lymphocytes with the arrival of foreign antigens. The T cells are activated to differentiate into effector and memory cells. 5

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DESCRIBING THE TCR COMPLEX The TCR : discovered in the 1980s, around the same time of structure of (MHC) discovered The antigen receptor of MHC-restricted T cells. a heterodimer consisting of two transmembrane polypeptide chains Each TCR two chain consists of : Ig-like N-terminal variable (V) domain, one Ig-like constant (C) domain, hydrophobic transmembrane region, short cytoplasmic region. The V regions of the TCR chains contain short stretches of amino acids where the variability between different TCRs is concentrated : (CDRs) 6

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CD3 and ? : noncovalently associated with the TCR heterodimer After TCR Ag recognition: transduce the signals that lead to T cell activation. 7

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Costimulators: The proliferation and differentiation of naive T cells: antigen-induced signals. signals from costimulators In the absence of costimulation : fail to respond and die by apoptosis or enter a state of anergy. Two signals for Effective T cell activation : signal 1: initiated by engagement of the TCR complex signal 2: initiated by binding of costimulatory receptors that transduce independent signals or enhance the signaling cascades initiated by TCR complex . 10

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Signal 1:Engagement of the TCR by peptide/MHC results in activation of multiple second messenger pathways. These signals by themselves, however, are not sufficient to stimulate T cell proliferation or differentiation into effector cells. Signal2: When signal 1 is delivered concurrently with a costimulatory signal (signal 2), instead of being rendered anergic, T cells are stimulated to expand and develop effector function 11

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The B7:CD28 Family of Costimulators: CD28: The best characterized costimulatory pathway in T cell activation . binds the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) expressed on activated APCs. iduces T cell proliferation by upregulating transcription of IL-2 and anti –apoptotic BCl-Xl. This outcome of CD28 signaling is not unique to the Il2 gene, significantly enhance mRNA half-life of multiple other cytokine genes. B7-1 and B7-2 : structurally similar integral membrane single-chain glycoproteins with two extracellular immunoglobulin (Ig)–like domains on the cell surface, B7-1 exists as a dimer and B7-2 as a monomer absent or expressed at low levels on resting APCs and are induced by microbial products that engage TLRs and cytokines such as IFN-? 13

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Costimulation of T cells by CD28 activates numerous signaling events. this signaling pathway by activation of the phosphatidyl inositol-3 kinase (PI3K) pathway that leads to increased enzymatic activity of AKT, a serine kinase that promotes cellular survival and glucose metabolism. CD28 engagement potentiates cytokine production both by inducing mRNA stability and by stimulating transcription through unique promoter elements (e.g., the CD28 response element [CD28RE]). also, CD28 stimulation results in activation of c-Jun kinase and several protein arginine methyltransferases. 14

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Costimulation of CD28 and the CD3 subunit of the TCR complex in CD4+ or CD8+ T cells triggers a complex series of membrane proximal signaling events that culminate in activation of the I?B kinase (IKK) complex. Free c-Rel translocates to the nucleus and binds to the CD28-responsive region of genes such as Il2, leading to more transcription. IL-2 is a mitogenic factor for T cells and promotes T cell proliferation. The TCR-CD28 pathway can also induce expression of Peli1. Peli1 can cause degradation and its inhibits the induction of cytokines such as IL-2 and results in a state of unresponsiveness known as 'T cell anergy' that is associated with peripheral T cell tolerance. IL-2R, receptor for IL-2. 16

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receptors homologous to CD28 ICOS: (inducible costimulator), CD278 It’s located on chromosome 2 is a disulfide-linked homodimeric glycoprotein. like CD28, contains a binding site for the p85 subunit of PI3K in its cytoplasmic domain. plays an essential role in T cell–dependent antibody responses, particularly in the germinal center reaction. When engaged: stimulates greater PI3K activity than does CD28, but some of the other CD28 effectors (e.g., Ras and Jun kinase) are not activated upon the binding of ICOS to its ligand. ICOS expression is low on naïve cells but up-regulated on cells stimulated via their TCR , Its ligand, called ICOS-L (CD275), is expressed on dendritic cells, B cells, and other cell populations. 17

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The inhibitory receptors of the CD28: CTLA-4(cytotoxic T lymphocyte antigen 4) PD-1 (programmed death 1). CTLA-4 present on activated T cells, competes for binding to B7-1 and B7-2 precluding CD28-mediated activating signals. CD28 and CTLA-4 provide two receptors that recognize the same ligands (the B7 molecules) but have opposite functional effects on T cell activation. CTLA-4: a high-affinity receptor for B7 CD28 has a 20- to 50-fold lower affinity for B7, and it may be engaged when B7 levels are relatively high engaged when B7 levels on APCs are low. 18

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Engagement of CTLA4 also induces inhibitory signals, including : changes in the spectrum of MAPKs that are activated and recruitment of phosphatases that counteract the effects of the TCR-stimulated PTKs. Failed costimulation in the setting of continued TCR signals may lead to apoptosis or anergy. 19

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The interaction of CD40L on T cells with CD40 on APCs enhances T cell responses by activating the APCs. CD40 ligand (CD40L) : a TNF superfamily membrane protein that is expressed primarily on activated T cells, and CD40 is a member of the TNF receptor superfamily expressed on B cells, macrophages, and dendritic cells. Functions of CD40: in activating macrophages in cell mediated immunity and activating B cells in humoral immune responses. Activated helper T cells express CD40L, which engages CD40 on the APCs and activates the APCs to make them more potent by enhancing their expression of B7 molecules and secretion of cytokines such as IL-12 that promote T cell differentiation. 20

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Antigen recognition by T cells together with costimulation induces the expression of CD40L on the activated T cells. CD40L engages CD40 on APCs and may stimulate the expression of B7 molecules and the secretion of cytokines that activate T cells. 21

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Signaling pathways: Signal transduction: refers to the intracellular biochemical responses of cells after the binding of ligands to specific receptors. Signaling initiated by receptors typically involves an initial cytosolic phase when the receptor or proteins that interact with the receptor may be post translationally modified. Then leads to the activation or nuclear translocation of transcription factors that are silent in resting cells, followed by a nuclear phase when transcription factors orchestrate changes in gene expression. 22

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PROXIMAL SIGNALING COMPLEX: The earliest step in intracellular signaling following TCR ligation is the activation of Src (Lck and Fyn) PTKs, leading to phosphorylation of the CD3 ITAMs. Recruitment of ZAP- 70 follows, leading to a cascade of phosphorylation events. Among the most important of the ZAP-70 targets are the transmembrane adapter protein linker for the activation of T cells (LAT) and the cytosolic adapter protein Src homology 2 (SH2) domain–containing leukocyte phosphoprotein of 76 kDa (SLP-76). LAT contains nine tyrosines that are phosphorylated uponTCR engagement, which bind the C-terminal SH2 domain of PLC?1, the p85 subunit of phosphoinositide 3-kinase (PI3K), and the adapters growth factor receptor-bound protein 2 (GRB2) and GRB2-related adapter downstream of Shc (Gads). 24

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PLC?1 ACTIVATION AND SIGNAL TRANSDUCTION: PLC?1 is bound to SLP- 76, Vav1, and LAT, where it is phosphorylated and activated by Itk. Activated PLC?1 then hydrolyzes the membrane lipid PIP2, producing the second messengers IP3 and DAG. Following TCR ligation, Itk is recruited to the membrane through PH domain interactions with PIP3, which has been locally generated by Lck-induced PI3K activity. At the membrane, Lck phosphorylates Itk, and the SH2 and SH3 domains of Itk interact with phosphorylated tyrosine 145 and the PRR of SLP-76, respectively . A second Tec family kinase, Rlk, can also phosphorylate PLC?1, resulting in a relatively mild defect in Itk-deficient mice and requiring the study of Rlk/Itk double-deficient mice to better understand the role of Tec kinases in T cell activation. 25

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DAG-MEDIATED SIGNALING PATHWAYS: results in the activation of two major pathway involving: Ras and PKC?. Ras is a guanine nucleotide– binding protein and is required for the activation of the serine-threonine kinase Raf-1, which initiates a mitogen-associated protein kinase (MAPK) phosphorylation and activation cascade. Raf-1 is a MAPK kinase kinase (MAPKKK) that phosphorylates and activates MAPKkinases (MAPKKs), which in turn phosphorylate and activate the MAPK’s extracellular signal-regulated kinase 1 (Erk1) and Erk2. Erk kinase activity results in the activation of the transcription factor Elk1, which contributes to the activation of the activator protein-1 (AP-1) ( Jun/Fos) transcription complex via regulation of Fos expression. Additionally, Erk activity can result in the transcriptional activation of signal transducer and activator of transcription 3 (STAT3) and in the serine phosphorylation of Lck. Ras is only active in the GTP-bound state, and its activation is facilitated by GEFs and is suppressed by GTPase-activating proteins (GAPs). Two Ras GEFs are present in T cells, son of sevenless (SOS) and Ras guanyl nucleotide-releasing protein (RasGRP). 26

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Ca2+-MEDIATED SIGNALING PATHWAYS: Ca2+ ions are universal second messengers in eukaryotic cells. The IP3 generated by TCR stimulated PLC?1 activity stimulates Ca2+- permeable ion channel receptors (IP3R) on the endoplasmic reticulum (ER) membrane, leading to the release of ER Ca2+ stores into the cytoplasm. Depletion of ER Ca2+ triggers a sustained influx of extracellular Ca2+ through the activation of plasma membrane Ca2+ release-activated Ca2+ (CRAC) channels in a process known as store-operated Ca2+ entry (SOCE). the sensor for depleted ER Ca2+ stores and the activator of CRAC channels as stromal interaction molecule (STIM) . STIM is an ER-resident transmembrane protein with a C-terminal cytoplasmic coiled-coil motif and, within the ER lumen, an N-terminal sterile a motif (SAM) and paired EF hands, where one hand binds a single Ca2+ ion with low affinity. Following ER Ca2+ depletion, STIM1 molecules aggregate in clusters that preferentially localize to sites of ER plasma membrane apposition, where they colocalize with clusters of Orai1. 27

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FUNCTIONAL RESPONSES OF T LYMPHOCYTES: The earliest responses changes in : expression of various surface molecules secretion of cytokines and the expression of cytokine receptors. Some of the functionally important molecules: CD69: increases after activation of T cell binds to and reduces surface expression of the sphingosine 1-phosphate receptor S1PR1 as a receptor that mediates egress of T cells from lymphoid organs. CD25 (IL-2Ra). The expression of this cytokine receptor enables activated T cells to respond to the growth promoting cytokine IL-2. CD40 ligand (CD40L, CD154). express high levels Within 24 to 48 hours after activation. The expression of CD40L enables activated T cells to mediate their key effector functions, which are to help macrophages and B cells. CTLA-4 (CD152) increases within 24 to 48 hours after activation. CTLA-4 earlier as a member of the CD28 family that functions as an inhibitor of T cell activation and thus as a regulator of the response. Adhesion molecules and chemokine receptors. reduce expression of L-selectin and the chemokine receptor CCR7 increase the expression of molecules that are involved in their migration to peripheral sites of infection and tissue injury such as the integrins LFA-1 and VLA-4 and various chemokine receptors 29

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IL-2 Secretion and IL-2 Receptor Expression: Secreted IL-2 is a 14- to 17-kD glycoprotein that folds into a globular protein containing four a helices. The most important cytokine produced by T cells early after activation, often within 2 to 4 hour after recognition of antigen and costimulators. growth, survival, and differentiation factor for T lymphocytes and major role in the regulation of T cell responses by virtue of its crucial role in the maintenance of regulatory T cells, so called T cell growth factor (TCGF). rapid and transient production , starting within 2 to 3 hours of T cell activation, peaking at about 8 to 12 hours, and declining by 24 hours. Functions of IL-2: stimulates the survival, proliferation, and differentiation of antigen-activated T cells. required for the survival and function of reg T cells 30

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Functional IL-2 receptors expression IL-2R: consists of three noncovalently associated proteins including IL-2Ra (CD25), IL-2/15Rß (CD122), and ?c (CD132). only IL-2Ra is unique to the IL-2R, without initiation of signal transduction,alone. IL-2/15Rß, contributes to IL-2 binding and engages JAK3-STAT5–dependent signal transduction pathways. common ? chain (?c): shared with IL-4, IL-7, IL-9, IL-15, and IL-21. The IL-2Rß?c complexes are expressed at low levels on resting T cells (and on NK cells) and bind IL-2 with a Kd of approximately 10-9 M. Cells that express IL-2Ra and form IL-2Raß?c complexes can bind IL-2 more tightly, with a Kd of approximately 10-11 M 31

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TH1 : Activated CD4 T cells generally secrete vast amounts of IL-2, although TH1 cells are believed to be the major source of this cytokine. Contrarily, a few studies reported increased survival of CD4 T cells in the absence of IL-2 signals during the contraction phase. This phenomenon might be explained by IL-2–mediated activation- induced cell death, whereby IL-2 is able to sensitize CD4 T cells for cell death through the proapoptotic Fas/Fas ligand pathway. TH2 : a requirement for IL-2 signaling during the priming phase for the induction and maintenance of IL-4Ra expression and TH2 lineage commitment of CD4 T cells. TH17 : IL-2 was able to inhibit generation of TH17 cells both in mice and human subjects by interfering with the transcription factor RORgt. T Reg : CD4 CD25 Treg cells express the transcription factor FoxP3, inducing upregulation of CD25 and other Treg cell– associated molecules, such as CTLA- 4 and the glucocorticoid-induced TNF receptor, and suppressing the production of IL-2, IFN-g, and IL-4 by effector T cells. 35

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Proliferation Clonal Expansion of T Cells: is mediated primarily by a combination of signals from the antigen receptor, costimulators, and IL-2. Before antigen exposure, the frequency of naive T cells specific for any antigen is 1 in 105 to 106 lymphocytes. After microbial antigen exposure, the frequency of all CD8+ T cells specific for that microbe may increase to about 1 in 3 to 1 in 10, representing a >50,000-fold expansion of antigen-specific CD8+ T cells, and the number of specific CD4+ cells increases to 1 in 100 to about 1 in 1000 lymphocytes 36

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Metabolism of T cell activation and differentiation: After T cells undergo clonal expansion: ferment glucose, even though with sufficient oxygen present to support mitochondrial oxidative phosphorylation This phenomenon is known as the Warburg effect :an unusual metabolic aspect of proliferating T cells and cancer cells. In contrast to proliferating T cells, quiescent T cells (i.e. nai¨ve and memory cells), like most normal cells, interchangeably breakdown glucose, amino acids, and lipids to catabolically fuel ATP generation .Quiescent cells can also use autophagy (the break down of intracellular components) to supply the molecules to fuel oxidative phosphorylation. quiescence is under active transcriptional control. TOB1 (transducer of ERBB2 1), LKLF (lung Kru¨ ppel-like factor) ,and FOXO (Forkhead box class O) transcription factors all have been suggested to promote quiescence in lymphocytes by actively maintaining the expression of inhibitors of cellular activation. 37

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Activated T cells have an anabolic metabolism where they maintain a high rate of nutrient uptake and build biomass at the expense of ATP. Glycolysis provides ATP for proliferating T cells while fatty acids and amino acids are incorporated into cellular components. By contrast, quiescent T cells have a catabolic metabolism where they use glucose, fatty acids, and amino acids for ATP generation through the TCA cycle and oxidative phosphorylation. Growth factor cytokines increase nutrient transporter expression and are important for cell survival, and in their absence, quiescent cells die of progressive atrophy. 38

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Differentiation of CD4+ T Cells: characterized by: ability to express surface molecules and to secrete cytokines that activate other cells. The signature cytokines : IFN-? for TH1 cells; IL-4, IL-5, and IL-13 for TH2 cells; IL-17 and IL-22 for TH17 cells. Differentiated from naive CD4+ T lymphocytes: mainly in response to cytokines present early during immune responses. differentiation involves transcriptional activation and epigenetic modification of cytokine genes. The process of differentiation, which is sometimes referred to as polarization of T cells. During the differentiation process, changes in chromatin structure occur through epigenetic mechanisms such as histone modifications, DNA methylation and generation of DNase I hypersensitive sites. 39

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CD4+ Th1 and Th2 subset differentiation, polarization and plasticity: Signal strength defined as the cumulative effect of peptide affinity for MHC class II, TCR activation, cytokines and costimulatory signals regulates CD4+ Th cell differentiation . Th1 polarizing conditions :synergistic TLR combinations and systemic type I IFNs. In addition, reciprocal signaling between CD40L upregulated on Th1 cells and CD40 on DCs enhances cross-priming and synergizes with CpG oligodeoxynucleotides (TLR9) and LPS (TLR4) for CD8+ CTL induction. Parasite, helminth and environmental allergens induce Th2 polarization and the production of IL-4 and IL-13 The IL-33 receptor is upregulated by the transcription factor GATA-3 and drives Th2 cytokine secretion. The IL-33 receptor is also essential for upregulating TGF-b and the induction of Th9 cells involved in allergic responses. Helminth antigens SEA and NES trigger DCs through TLR2, generating a muted DC activation phenotype. Th2-promoting DCs stimulated with TNFa, CT or TSLP display a higher CD86/CD80 ratio and increased expression of Jagged 1 and OX40L in comparison with Th1-promoting DCs that express Delta 4. In addition, bone marrowderived DCs stimulated with CT and house dust allergen upregulate cell surface expression of both cKIT and its ligand, SCF. Autocrine activation of the cKIT–PI3K–AKT pathway induces production of large amounts of IL-6, a critical regulator of Th2, Th17 and IL-10 producing Tr1 cells and an inhibitor of iTregs. Abbreviations: CD40L CD40 ligand; CT cholera toxin; DMA dust mite allergen; MALP-2 NES Nippostrongylus excretory secretory; SCF stem cell factor; SEA Schistomiasis egg antigen; TSLP thymic stromal lymphopoietin. 40

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TH1 Differentiation: TH1 differentiation : mainly by the cytokines IL-12 and IFN-? and occurs in response to microbes that activate DC, macrophages, and NK cells. IFN-? and IL-12 stimulate T-bet, STAT1, and STAT4. IFN-? activates the transcription factor STAT1, which in turn stimulates expression of T-bet. T-bet promotes IFN-? production through transcriptional activation of the IFN-? gene an inducing chromatin remodeling of the IFN-? locus. The ability of IFN-? to stimulate T-bet expression set up a positive amplification loop. IL-12 contributes to TH1 commitment by activating the STAT4, which further enhances IFN-? production. STAT1 and STAT4 also contribute to the regulation of Tbx21 (the gene that encodes T-bet) expression 42

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Hlx Th1-specific homeobox gene that interacts specifically with T-bet. Its detectable 3 days after Th1 differentiation and is restricted to Th1 but not Th2 T cell clones . Hlx and T-bet synergistically promote IFN-g expression when co-expressed Thus, as a cofactor for T-bet T-bet: rapidly and specifically induced in developing Th1 but not Th2 cells its expression appears to be controlled by both TCR and IFN-? R -STAT1 signals, but not by the IL-12-STAT4 pathway. STAT4: critical for IL-12 signaling and thus the full commitment of Th1 cells. it is dispensable for initial IFN-g expression. STAT4 act through binding to regulatory genomic regions of the IFN- ? gene or interacting with general transcriptional co-activators p300/CBP. 43

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TH2 Differentiation: stimulated by the cytokine IL-4 and occurs in response to helminths and allergens. In chronic T cell stimulation, often without the strong innate immune responses that are required for TH1 differentiation. IL-4 stimulates TH2 development by activating the transcription factor STAT6, and STAT6, together with TCR signals, induces expression of GATA-3. GATA-3 is a master regulator of TH2 differentiation, enhancing expression of the TH2 cytokine genes IL-4, IL-5, and IL-13, which are located in the same genetic locus. 44

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STAT6: STAT6, activated by IL-4 stimulation, is the major signal transducer in IL-4-mediated Th2 differentiation. In vitro, one of the mechanisms of STAT6 is to promote Th2 differentiation through inducing high levels of the transcription factor GATA-3. GATA-3: GATA-3 dictates Th2 differentiation by regulating Th2 cytokine production through binding to the IL-4 locus that encompasses the IL-4, IL-5 and IL-13 genes. Forced expression of GATA-3 in Th1 cells led to the induction of IL-4 production. c-Maf, IRF-4 and Gfi-1: c-Maf is an AP-1 family transcription factor selectively up-regulated in Th2 cells. It is required for the production of IL-4 but not other Th2 cytokines. IRF-4 promotes Th2 differentiation through up-regulating GATA-3. Gfi-1 is a transcription factor immediately induced following IL-4-stimulation and TCR stimulation. Gfi-1 appears to function by selecting GATA-3hi cells for growth by modulating both upstream and downstream events of IL-2 signaling. 45

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TH17 Differentiation: The development of TH17 cells is stimulated by proinflammatory cytokines produced in response to bacteria and fungi. Various bacteria and fungi act on dendritic cells and stimulate the production of cytokines including IL-6, IL-1, and IL-23. Engagement of the Dectin-1 on DC by fungal products is a signal for the production of these cytokines. The development of TH17 cells is dependent on ROR? t and STAT3: TGF-ß and IL-6 and IL-1, work cooperatively to induce the production of ROR?t, a member of the retinoic acid receptor family. IL-6, activate the STAT3, which functions with ROR?t to drive the TH17 response 46

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Epigenetics and Th1 cell differentiation: In the eukaryotic nucleus DNA is compacted by wrapping 146 bp of DNA around the four core histone proteins H3, H4, H2A, and H2B, which forms the basic structural unit of chromatin, the nucleosome. The N-terminal tails of histone proteins, modified such as acetylation, methylation, and phosphorylation . modifications have a direct effect on the gene expression by condensing or relaxing the chromatin Or indirect where the histone modifications act as signals that are recognized by transcription factors or cofactors. During the differentiation process, changes in chromatin structure occur through epigenetic mechanisms such as histone modifications,DNA methylation and generation of DNase I hypersensitive sites . Histone 3 lysine 4 trimethylation (H3K4me3) and H3K27me3 are the most commonly studied epigenetic modifications in T helper cell subtypes. The H3K4me3 modification generally centered around the transcription start site(promoter) of active genes In contrast, the H3K27me3 modification is associated with a repressive epigenetic environment. T-bet activates Th1 signature genes in part by recruiting chromatin remodeling complexes including Jmjd3, a Brg1- containing SWI/SNF-complex, and an H3K4me2-methyltransferase complex to its target genes 47

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Histone acetylation (H3Ac, H4Ac) associates with active transcription. Unmodified loci in euchromatin are targets of silencing by DNA methylation and the accrual of corepressive complexes. There is a good correlation between DNA CpG methylation and histone H3 lysine 9 (H3K9) marks in differentiated cells, highlighting cooperativity between these regulatory mechanisms. Nai¨ve CD4+ T cells are poised to express either Th1 or Th2 transcription factors T-bet and GATA-3, respectively, indicated by both permissive trimethylation of lysine 4 on histone 3 (H3K4me3) and repressive di- or trimethylation of lysine 27 (H3K27me3). These bivalent histone methylation marks persist in all CD4+ T cell lineages, suggesting the potential for re-expression 48

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Chromatin remodeling of the IFN-? locus Signals from IL-12R IFN-? chromatin remodeling complexes HAT Enhancers 49

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Chromatin remodeling of the Ifn-g locus controls expression in polarized CD4+ Th1 cells. The euchromatin in the Ifn-g locus of nai¨ve CD4+ T cells contains both permissive histone H3 and H4 bi- and tri-methylation marks and repressive H3K27 methylation characteristic of the bivalent state. Th1: T-bet binds proximal promoter and intronic enhancers and can activate transcription of IFN-? . Additional transcription factors recruit coactivator complexes and HATs such as p300/CBP that open chromatin, maximizing gene expression. T-bet directly induces the expression of both Runx- 3 and HLX, which enhance activity at the IFN-? promoter. Signals delivered through the IL-12 receptor and IFN-? receptor activate STAT-4 to enhancers and recruit ATP dependent chromatin remodeling complexes, SWI/SNF and BRG-1. Additionally, signaling through the ? common chain cytokine receptor by IL-2 or IL-15 is necessary for Jak3/STAT-5 activation to promote the accessibility of the promoter for T-bet binding. Th2: increased methylation of an intronic enhancer and a -53base pair cis regulatory region prevent ATF2/CREB binding to the promoter. Ikaros binds the IFN-? locus, recruiting a corepressor complex and DNA methylation enzymes and also silences the T-bet locus, in turn silencing transcription of Ifn-g. LSD1 removes permissive histone marks, recruiting DNMT3a and DNMT3b, which methylate CpG regions. Methyl DNA binding proteins recruit the corepressor complex to the methylated CpGs, whereas HDACs remove acetyl groups on lysine tails. HP1 condenses the locus into inactive heterochromatin. Finally, repressive histone marks are added (trimethylation of H3K27 and H3K9). Abbreviations: BRG-1 Brahma-related gene 1; HAT histone acetyltransferase; HP1 histone protein 1; LSD1 lysine specific demethylase 1; SWI Switch; SNF sucrose nonfermentor. 50

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T-bet activates Th1 signature genes, including IFN-?, during Th1 cell development by functionally regulating multiple aspects of chromatin remodeling. In nai¨ve CD4+ T cell, the nucleosomes encompassing the IFN-? locus are marked by repressive H3K27me3 epigenetic modifications to keep the locus in a compacted chromatin state. As nai¨ve CD4+ T cells begin to express T-bet and differentiate toward the Th1 phenotype, T-bet physically recruits an H3K4me2-methyltranserase complex containing Set7/9, the H3K27-demethylase Jmjd3, and a SWI/SNF-general chromatin remodeling complex through its association with Jmjd3. Collectively, the T-bet dependent remodeling events create a more permissive or accessible chromatin environment, which allows for the binding of additional transcriptional regulatory proteins, including STAT4, to activate gene transcription. 51

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Differentiation of CD8+ T Cells into Cytotoxic T Lymphocytes The full activation of naive CD8+ T cells and their differentiation into functional CTLs and memory cells may require the participation of CD4+ helper cells. The induction of effector CD8+ T cells is dependent on many of the same classes of signals as for effector CD4+ T cells, antigenic peptide/MHC, pathogen-related inflammatory signals including costimulators, and cytokine signals. Helper T cells may secrete cytokines that stimulate the differentiation of CD8+ T cells. Antigen-stimulated helper T cells express CD40 ligand (CD40L), which binds to CD40 on APCs and activates (“licenses”) these APCs to make them more efficient at stimulating the differentiation of CD8+ T cells. The most specific feature of CTL differentiation: development of perforin and granzymes, Two transcription factors T-bet and eomesodermin 52

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models of T CD8 Activation: Programming model Environmental model 53

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models of T CD8 Activation: (a) Programming model. CD4+ help is required during the primary CD8+ T cell response to antigen. Although CD4+ T cells are dispensable for production of CD8+ effector T cells, CD4+ T cell help during priming imprints CD8+ T cells with the ability to differentiate into useful memory cells. Helped CD8+memory T cell pools show more potent functional reactivity, prolonged maintenance and the ability to control pathogens in a recall response. (b) Environmental model. the effect of CD4+ T cells is mediated later in the CD8+ response. Here, CD4+ T cells provide a suitable environment for the preservation of CD8+ memory function and persistence. In this model, CD8+ T cells are not irreversibly programmed by CD4+ help. Instead, helped and unhelped CD8+ memory T cells can interconvert, depending on the availability of bystander CD4+ T cells, throughout the life of the CD8+memory T cell. 54

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Development of Memory T Cells: Immune memory indicates a qualitatively and/or quantitatively distinct immune response upon successive (but interrupted) exposures to antigen. memory T cells have several key features that distinguish them from naïve cells: much higher frequencies than naïve T cells self-renewal as do hematopoietic stem cells memory cells undergo intermittent cell division once every 2–3 wk show enhanced ability of Ag-independent self-renewal IL-2, IL-7, IL-15, and IL-21 rapid clonal proliferation functional activation 55

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Following infection, T cells undergo massive proliferation while differentiating into effector cells. Responses typically peak approximately 7–10 days after infection and most cells die off within the next few weeks leaving behind 5–10% to become long-lived memory cells. evidence supports that antigen specific CD8+ T cell memory represents a stem cell-like state can differentiate into effector and memory cell populations in response to antigen stimulation. CD8+ memory is characterized by the expression of CD161, IL-18R and the stem cell markers c-kit,Bcl-2,Bcl-xL, CD28 and CD127 (IL-7 receptor). sustained signal strength during the primary response is necessary to generateCD4+Tcell memory. CD4+ memory cells are derived from effector cells through a process of linear differentiation and represent a quiescent population with limited self-renewal characteristics. 56

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TCR signalling intensity and memory CD4 T cell development: nai¨ve CD8+ T cells can be easily triggered into a programmed course of differentiation with a relatively brief period of stimulation. nai¨ve CD4+ T cells require prolonged antigenic stimulation for differentiate into effector and memory cells. nai¨ve CD4+ T cells require a high threshold of TCR signalling in order to fully differentiate into effector cells that can convert to memory cells. the presence of an unphysiologically high precursor frequency of antigen (Ag)-specific nai¨ve CD4+ T cells during the primary response is detrimental for memory formation. 57

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Linear versus non-linear differentiation of memory CD4+ T cells: There are two competing models of memory T cell development: linear divergent differentiation Linear: activated nai¨ve T cells develop initially into cytokine-producing effector cells before a small fraction of these cells convert to memory cells. divergent differentiation: a fraction of activated nai¨ve T cells differentiate directly into memory cells, bypassing the effector phase. 58

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Common markers used for T-cell memory. 59

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Naive T cells CD45RA+ CCR7 + (lymph node homing) CD28 + (costimulatory receptors) CD27 + (costimulatory receptors) lack of expression of cytolytic molecules Granzymes perforin CD107a 61

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Two broad categories of memory cells: effector-memory (Tem) : being CD62Llow, CCR7low central-memory (Tcm): CD62Lhi, CCR7hi Effector memory T (TEM) cells are terminally differentiated and acquire effector function immediately after re-stimulation central memory T (TCM) cells have a longer lifespan and can differentiate into TEM cells following antigenic challenge. 62

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short-lived effector memory T cells (TEM) CCR7- migrate to peripheral tissues (CCR3 + & CCR5+) CD45RA-/+ / CD45RO‡ CD27-/+ /CD28-/+ become effectors more rapidly (cytotoxicity and IFN-? secretion) reduced ability of: proliferation secretion IL-2 TEM cells express homing receptors that facilitate migration to nonlymphoid sites of inflammation and produce a variety of cytokines, including IFN-?, IL-4 and IL-5, within several hours of TCR stimulation. 63

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Long-lived central memory T cells (TCM) CD62L+ & CCR7 + Homing to lymph nodes CD45RA- / CD45RO‡ rapidly differentiate into cells endowed with effector functions upon exposure to Ag CD40L+++ Proliferation +++ Secretion IL-2 +++ TCM cells do not produce any of the prototypic cytokines of the effector cell lineage immediately after stimulation through the TCR, although they do secrete IL-2 and proliferate extensively and acquire effector lymphokine production later. These cells express CD62L (L-selectin) and the chemokine receptor CCR7. 64

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Embracing Diversity: (A) The chart shows the relative longevity and recall expansion potential of ‘‘idealized’’ naive, effector, Tcm, and Tem cell populations, as they are typically discussed, as well as conflicting models of lineage differentiation. (B) The shaded area indicates a distribution ‘‘cloud’’ of cells with effector-through-memory-like properties. Many populations in this cloud would typically be designated ‘‘Tem,’’ but as indicated, there is also diversity among the ‘‘Tcm’’ cell pool. Shown are memory populations identified in various tissue sites, including Tem cells from lung bronchoalveolar lavage (BAL) and small intestine (SI) and Tem and Tcm cells isolated from spleen at different memory time points (‘‘Early’’ and ‘‘Late’’ Tcm and Tem) . CD8+ T cells primed without sufficient CD4+ T cell help (‘‘Helpless’’), memory-like cells made through lymphopenia- driven homeostatic proliferation (‘‘HP’’ memory cells), T cells driven to ‘‘exhaustion’’ by chronic antigen exposure (‘‘Tex’’), and effector cells maintained into the long-lived phase (‘‘Persistent effector’’). The position of CD8+ memory T cells produced in IL-15- (or IL-15Ra-) deficient mice, and of CD25-deficient CD8+ memory T cells is also illustrated, though it is not yet clear whether such cells are representative of populations generated in natural responses (hence, these cells fall outside the ‘‘cloud’’). Relative positions of populations are not intended to be precise but merely to indicate the diversity of groups 66

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Stem-cell-like memory cells have physical characteristics of very young immune cells. They still have the potential to differentiate and become many different types of immune cells, making them extremely valuable. 67

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Immunological memory is thought to depend on a stem cell–like, self-renewing population that has an enhanced capacity for self-renewal and a multipotent ability to derive central memory, effector memory and effector T cells. These cells, found within a CD45RO-, CCR7+, CD45RA+, CD62L+, CD27+, CD28+ and IL-7Ra+ T cell compartment characteristic of naive T cells. However, they expressed CD95, IL-2Rß, CXCR3, and LFA-1, and showed numerous functional attributes distinctive of memory cells. Compared with known memory populations, these lymphocytes had increased proliferative capacity. flow cytometric analysis of human peripheral blood identified naive-like CD4+ and CD8+ T cells with the CD95+CD122+ phenotype and high levels of expression of BCL-2 and CXCR3. Like TCM and TEM cells, human TSCM cells were found to have undergone clonal expansion and to be long-lived, and they rapidly acquired effector function following T cell receptor (TCR) stimulation. The gene expression pattern of TSCM cells indicated that they are a distinct population of memory T cells that are less differentiated than TCM and TEM cells. 68

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Simultaneous generation of TEM and TCM cells. first pathway (I), effector cells successfully enter the memory phase as TEM cells. second pathway (II), effector cells lack the potential to enter the memory phase and die. third pathway (III), cells present during the effector phase (TFH cells or TCM cells) become central memory cells after interacting with B cells. A model for the generation of TEM and TCM cells: 69

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pathway I: Naive T cells are first stimulated by the presentation of pMHCII by dendritic cells and then proliferate to form effector cells under the influence of cytokines from the innate immune system. Some of these early effector cells, perhaps those that do not interact with B cells, commit to a particular lineage (such as TH1), and some of these survive to become lineage-committed TEM cells pathway II: Treg cells may also be induced but fail to survive or change into TH1 effector memory cells pathway III: Other early effector T cells interact with B cells and receive signals through ICOS. Some of these cells become TFH effector cells that survive as long as antigen and pMHCII-presenting germinal center B cells are present. After the germinal center reaction ends, some of the TFH cells may become quiescent memory cells while retaining low expression of some TFH cell markers such as CXCR5 and losing expression of others such as PD-1. In this case, the TCM cells without lineage commitment, such as the CXCR5+ memory cells observed in several systems, are the descendants of TFH cells. Alternatively, some early effector cells may become CXCR5+ TFH cells and others may become CXCR5+ TCM cell precursors, both in response to ICOSL signals from B cells. 70

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evidence available at present indicates that committed TH1, TH2 and perhaps TH17 effector cells survive the contraction phase to form TEM cells. Other evidence indicates B cells are important drivers of the TEM-TCM ‘decision’. However, the big question of why only 10% of the effector cells become memory cells remains unresolved. 71

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Bcl-6 and Blimp-1 Bcl-6 and Blimp-1 (encoded by Prdm1) are transcriptional repressors with the ability to block each other’s expression Bcl-6 and Blimp-1 were previously : critical regulators of effector and memory differentiation of B lymphocytes. Bcl-6 and Blimp-1 : one of the key transcriptional regulators of effector and memory differentiation in CD4+ T and CD8+ T cells. Bcl-6 and Blimp-1 regulatory axis as a ubiquitous mechanism for controlling effector and memory lymphocyte differentiation and function. 72

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Lymphocytes with higher expression of Bcl-6 exhibit greater proliferative capacity but less secretory capacity. Lymphocytes with higher expression of Blimp-1 exhibit lower proliferative capacity and greater secretory capacity. Although exhausted CD8+ T cells have large amounts of Blimp-1 and lower proliferative capacity, they do not have more secreted effectors. 75

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Homeostasis of memory CD4+ T cells: signalling from a combination of IL-7 and IL-15, but not MHC-I, regulates homeostasis of memory CD8+ T cells, the factors controlling homeostasis of the memory CD4+ T cell pool are more controversial. IL-7 appears to have a bigger role than IL-15 in supporting. survival of memory CD4+ cells. IL-15 has a less prominent role for memory CD4+ cells than for NK and memory CD8+ cells, these latter cells being much more reliant on IL-15 than IL-7 for their homeostasis. This difference in IL-15 dependence correlates closely with the expression levels of the IL-15 receptor, CD122, which is displayed at much lower levels on memory CD4+ cells than on NK and memory CD8+ cells. 76

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Role of apoptotic proteins in memory T cell survival: A delicate balance of pro-apoptotic and anti-apoptotic molecules dictates survival of T cells. Both IL-7 and IL-15 have been shown to support T cell survival by increasing the expression of anti-apoptotic molecules, such as Bcl-2 and Mcl-1. the proapoptotic BH3-only molecule Bim has been implicated in effector cell death during the contraction phase. however, Bim memory CD4+ cells did not have an overtly extended lifespan. In addition, high levels of Bim expression were found on effector CD4+ T cells derived from suboptimally activated nai¨ve CD4+ T cells, that is, cells that failed to develop into memory cells. Bcl-2 has a prominent role in controlling the survival of memory CD4+ T cells. 78

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survival of human memoryCD4+Tcells is mediated partly by inhibiting the transcription of Bim and Fas via: inactivation of forkhead box O3a (FOXO3a) transcription factor with activation of STAT5a. Another BH3-only pro-apoptotic molecule, Noxa, play an intermediary role in controlling memory CD4+ T cell generation. And, Bmi1 controls Noxa expression. indicating that Bmi1 promotes memory CD4+ cell generation through repression of the Noxa gene. 79

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Recall response of memory T cells: Rapid recall immune responses by memory T cells are attributed to their increased precursor frequency of Ag-specific cells plus their heightened responsiveness to Ag. major reasons for increased sensitivity to Ag reduced co-stimulatory requirements for activation of memory T cells compared to nai¨ve T cells. elevated expression of the TCR proximal tyrosine kinase, Zap70, in memory CD4+ T cells. By the way, recent studies reveals that nai¨ve and memory CD4+ T cells commence cell proliferation at a similar time point after Ag exposure and undergo a similar rate of cell division. 80

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