1、上堂课回顾：,B淋巴细胞的成熟：,祖B细胞，前B细胞，非成熟B细胞，成熟B细胞 两次选择： 阴性选择：在骨髓中，表达与自身抗原有强亲和力的抗原受体的非成熟的B细胞将无法成为成熟的B淋巴细胞。 阳性选择：与T淋巴细胞的阳性选择不同，有可能，只要能够表达抗原受体识别抗原的B细胞都能够被保留下来。,T淋巴细胞的成熟：,祖T细胞，前T细胞，双阳性阶段，单阳性阶段，成熟阶段两次选择：1. 阳性选择：,2. 阴性选择：,Chapter 8 Activation of T lymphocytes,How to activate the T cell?,In peripheral lymphoid organ
2、s, Nave T lymphocytes recognize antigens and are activated. In lymphoid organs or in nonlymphoid tissues, effector T cells recognize antigens and are activated. The activation of T cells requires a. antigen presented by APCb. costimulatorsc. cytokines produced by the APCs and by the T cells. 4a. Nav
3、e T cells require activation by dendritic cells;4b. Effector T cells can respond to antigens presented by a wider variety of APC.,What the activated T cells can do?,Proliferation Differentiation into Effector Cells CD4+ T cell: Th1 and Th2 Th1: Stimulate cytokine( IL-12 and IFN-g), secrete cytokines
4、: IL-2, INF-g and TNF-b. Th2: Stimulate cytokine:IL-4, secrete cytokines: IL-4,5,6,10,13. CD8+ T cell differentiate into functional CTL. 3. Differentiation into Memory Cells 4. Decline of T Cell Responses,Role of Costimulators in T Cell Activation,Costimulators :CD28 : B7-1 and B7-2 The expression o
5、f costimulators is regulated to make sure the correct time and place expression The expression of B7 costimulators is increased 2a. by microbial products, 2b. by cytokines of innate immunity,3. Mature dendritic cells express the highest levels of costimulators. 4. Adjuvants are products of microbes
6、that stimulate the expression of costimulators. 5. Previously activated effector and memory T cells are less dependent on costimulation by the B7:CD28 pathway than are nave cells.,Role of CD40 in T cell activation,Activated T cells express CD40L APCs express CD40 CD40L binding to CD40 can deliver si
7、gnals that enhance the expression of B7 costimulators on the APCs.Secretion of cytokines IL-12 is increased upon CD40L interaction with CD40.,Signal Transduction by the TCR Complex,1. What is the goal : activate the transcription of genes that are silent in nave cells, these proteins mediate the res
8、ponses and functions of activated T cells.,2. How is signal transduction triggered: by ligation of multiple antigen receptors and coreceptors. 3. What happened after signal transduction in cell: clustering of membrane receptors, tyrosine phosphorylation of several protein, and recruitment and activa
9、tion of adapter proteins.,4. What the important biochemical pathways in the activation of T cells: 1) Ras-MAP kinase pathway2) the protein kinase C pathway,Major steps in signaling by the TCR,Early membrane events:,1. Formation of the Immunological Synapse:,Immunological synapse: the region of physi
10、cal contact between the T cells and the APC T cell components participated in are : TCR complex, coreceptors(CD4 or CD8 ), CD28, the enzymes and adaptor proteins. The formation is triggered by antigen recognition 4) Brings signaling molecules into proximity to one another,The formation of synapse wa
11、s demonstrated by confocal microscopy.,2. Activation of Tyrosine Kinases,Tyrosines residues in: CD3, z chain. Tyrosine kinases: Lck, that is associated with the cytoplasmic tails of CD4 and CD8. Fyn , another tyrosine kinase in physical association with the CD3 molecules. ZAP-70, attached to the tyr
12、osine phosphorylated ITAMs z chain. ZAP-70 is only expressed in T and NK cells.,2. Activation of Tyrosine Kinases,Procedure: Lck is activated by autophosphorylation, then the active Lck phosphorylates the tyrosines in the ITAM of the CD3 and the z chain ZAP-70 binding to two phosphorylated tyrosine
13、residues of each ITAMs in the z chain Lck phosphorylates the tyrosines of ZAP-70. ZAP-70 acquires its own tyrosine kinase activity. Multiple ZAP-70 could be recruited to the multiple phosphorylated ITAMs.,3. Recruitment and Activation of Adapter Proteins,Adapter proteins bring the signaling molecule
14、s into specific cellular compartments. Can bind other proteins. ZAP-70 phosphorylated tyrosines of LAT. Activated LAT binds phospholipase Cg 1, SLP-76, and Grb-2. Serves to bring a variety of downstream components of TCR signaling pathways close to their upstream activators.,Ras-MAP Kinase Signaling
15、 Pathways in T lymphocytes,Proteins that were needed in the pathway: 1. Ras: a member of a family of 21-kD guanine nucleotide-binding proteins, ( small G protein) has a lot of activation responses in different cell types. The reason is that Ras can recruit or activate various cellular enzymes. 2. MA
16、P kinases: ERK, extracellular receptor-activated kinase JNK, c-Jun N-terminal kinase also called stress-activated protein. P38,Ras-MAP Kinase Signaling Pathways in T lymphocytes,LAT can bind Grb-2, then the Grb-2 recruits the Ras GTP/GDP exchange factor -Sos ( son of sevenless) Sos catalyzes GTP for
17、 GDP exchange on Ras. Ras.GTP activates mitogen activated protein ( MAP ) kinases -ERK The ERK phosphorylates a protein called ELK, and stimulates transcription of Fos, Two other MAP kinases: Rac.GTP activates JNK, JNK phosphorylates c-Jun, another component of transcription factor. p38, is too acti
18、vated by Rac.GTP and in turn activates various transcription factor.,Pathway:,Calcium- and protein Kinase C-Mediated Signaling Pathways in T lymphocytes,PLCg1 is recruited to phosphorylated LAT, PLCg1 is a cytosolic enzyme specific for inositol（肌糖） phospholipids（磷脂质）. PLCg1 is phosphorylated by ZAP-
19、70. Phosphorylated PLCg1catalyzes the hydrolysis of a plasma membrane phospholipid called PIP2（磷脂酰肌醇二磷酸）, generating two products: IP3（三磷酸肌醇） and DAG（二酰基甘油）.,Signal pathway of IP3: IP3 binds to the receptor of ER and stimulates release of membrane-sequestered calcium stores. Calcium ion concentratio
20、n increased, and binding to a calcium-dependent regulatory protein called calmodulin（钙调蛋白）. Calcium-calmodulin complexes activated calcineurin（钙调磷酸酶） that is important for transcription factor.,Calcium- and protein Kinase C-Mediated Signaling Pathways in T lymphocytes,Calcium- and protein Kinase C-M
21、ediated Signaling Pathways in T lymphocytes,DAG is hydrophobic, and remains in the membrane where it is formed. The elevated free cytosolic calcium and DAG activates membrane-associated PKC（蛋白激酶C）. PKC q isoform localizes to the immunological synapse. PKC q activates the nuclear translocation of the
22、 NF-kB transcription factor.,Signal pathway of DAG:,Activation of Transcription Factors that Regulate T Cell Gene Expression,The requirement for multiple transcription factors accounts for the need to activate multiple signal transduction pathways. For example IL-2: three factors( NFAT, AP-1, NF kB)
23、are needed, and all the sites must be occupied for maximal transcription of IL-2 gene. These three factors are critical for most T cell responses.,Activation of Transcription Factors that Regulate T Cell Gene Expression,NFAT: A transcription factor required for the expression of IL-2, IL-4 and other
24、 cytokines. Four types of NFAT, NFAT1 and 2 in T cells. In resting T cells, NFAT presents in an inactive, serine phosphorylated form in the cytoplasm. It is activated by the calcium-calmodulin-dependent phosphatase calcineurin. Calcineurin dephosphorylates cytoplasmic NFAT, uncovering a nuclear loca
25、lization signal.,Activation of Transcription Factors that Regulate T Cell Gene Expression,2. AP-1,A transcription factor found in many cell types, and is specifically activated in T lymphocytes by TCR mediated signals. It is composed of Fos and Jun. From the Erk and PKC pathway, Fos gene was generat
26、ed. JNK phosphorylates c-Jun, Fos and phosphorylated c-Jun consisted AP-1.,Activation of Transcription Factors that Regulate T Cell Gene Expression,3. NF kB,A transcription factor that is activated in response to TCR signals and is essential for cytokine synthesis.In resting T cells, NF kB is presen
27、t in the cytoplasm in a complex with a inhibitors IkB. After phosphorylation of IkB, ubiquitin attach to the protein and be degraded. NF kB is released and translocated to the nucleus.,抗原MHC复合体,T淋巴细胞激活：,共刺激信号,细胞因子,T淋巴细胞被激活,佐剂,免疫突触形成,T细胞内信号转导激活,T细胞内信号转导激活：,CD4 分子上Lck 自身磷酸化,CD3分子ITAM酪氨酸基序磷酸化,ZAP70结合,Z
28、AP70被磷酸化并且酪氨酸激酶活性激活,多个ZAP聚集,LAT蛋白被磷酸化,LAT蛋白被磷酸化,磷脂酶Cg1活化,Grb-2活化,PIP2,IP3,DAG,PKC,NFkB,NFAT,ERK,ELK,Fos,JNK,JUN,AP-1,Biochemistry of Costimulation,CD28 can activate MAP kinase pathway, including ERK and Jun. Can activate NFkB to bind to IL-2 gene promoter. May also block inhibitory signals in T cell
29、s. CTLA-4 blocks normal phosphorylation of TCR associated z chain. Not known 1. How is this choice made?,Known,One possibility:,CTLA-4 binds to B7-1 and B7-2 with 50-fold higher affinity than does CD28.,Altered peptide Ligands:,The TCR on an individual T cell contact only a few amino acid residues o
30、f a particular MHC-bound peptide.,2. If one or two TCR contact residues have been changed, the responses will be different from the native peptide.2a: may induce a subset of the functional responses.2b: others deliver negative signals to specific T cells that inhibit responses to native peptides.,P2( Tyr53), P5(Leu56), P8( Asn59), P10(Arg61) and P11(Trp62),3. APLs: Peptides with altered TCR contact residues that elicit different responses4. Utilization: microbes produce altered versions that inhibit host immune responses.,
