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T-Cell Maturation, Activation, And Differentiation

T-Cell Maturation, Activation, And Differentiation. W. Robert Fleischmann, Ph.D. Department of Urologic Surgery University of Minnesota Medical School rfleisch@umn.edu (612) 626-5034. Objectives. To understand T cell maturation in the thymus, including positive and negative selection

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T-Cell Maturation, Activation, And Differentiation

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  1. T-Cell Maturation, Activation, And Differentiation W. Robert Fleischmann, Ph.D. Department of Urologic Surgery University of Minnesota Medical School rfleisch@umn.edu (612) 626-5034

  2. Objectives • To understand T cell maturation in the thymus, including positive and negative selection • To understand T cell activation, including signals initiated by antigen recognition and by costimulatory signaling • To understand T cell differentiation and the generation of the various subsets of T cells

  3. James Cormack, age 22 months, is brought to your clinic by State authorities. He was picked up from a day-care center following a report of possible child abuse. The examining physician notes that James has multiple severe bruises on his body. James’s family has not yet been contacted by the State juvenile authorities, so no medical history is available. The physician notes that James has a stuffy nose. An examination indicates that James has a purulent discharge from his nose, consistent with sinusitis. James also appears to have eczema on his trunk. The physician requests a complete WBC and sends a sample of the discharge for culture.

  4. Complete WBC results: Total WBC counts: 12,000/µl (4,100-10,900/µl) Differential WBC counts*: Neutrophils 35% (22-40%) Lymphocytes 60% (49-73%) Monocytes 5% (3-10%) Eosinophils 0% (0-6%) Basophils 0% (0-3%) RBC parameters: all normal Partial thromoplastin time 23 sec (18-28 sec) Prothrombin time 13 sec (12-14 sec) Platelet count 30,000/µl (140,000-450,000/µl) What do these results suggest? *Values for a patient who is 2 weeks to 4 years old.

  5. Using the telephone number provided by the day-care center, James’s mother is contacted. She arrives at the hospital and is appalled and upset to learn that James had been taken into custody by State authorities and transported to the hospital without her and her husband’s knowledge. Once she is sufficiently calm, she provides the physician with James’s social and medical history. She indicates that she and James have moved to the U.S. from Ireland two weeks ago, catching up with her husband who began a post-doctoral fellowship at the local university a month earlier. She dropped James off at a local church’s day-care center and went job hunting.

  6. She indicates that James has had a history of bruising and says that it seems to be getting worse lately. However, she thinks his bruising is due to the combination of his greater level of activity and increasingly risky behavior (climbing on things and running out of control into furniture). When asked, she also mentions that James has a number of ear and sinus infections. Several months ago, he was diagnosed with pneumonia. She doesn’t know what bacteria have been responsible for these infections. In response to a specific question, she indicates that James has been staying at home with her or with his grandparents. He has not been at a day care center. What do these results suggest? What would you do next?

  7. Number of B cells and T cells in peripheral blood: T cells 80% (60-95%) CD4+ 54% (60-75%) CD8+ 26% (25-30%) NK cells 10% (4-30%) B cells 10% (4-25%) Response to T cell mitogens: Concanavalin A: normal response Phytohemagglutinin: normal response Candida albicans: no response Response to B cell mitogen Pokeweed mitogen: normal response Immunoglobulin levels in blood: Serum IgG 350 mg/100 ml 350-800 mg/100 ml Serum IgA 40 mg/100 ml 15-50 mg/100 ml Serum IgM 13 mg/100 ml 25-75 mg/100 ml What do these results suggest?

  8. Culture results from nasal swab: Streptococcus pneumoniae Mean volume of platelets: <5 fL (7.5-11.5 fL) What do these results suggest?

  9. T Cell Maturation

  10. T Cell Maturation • B cells are mature when they leave the bone marrow. • T cells require “higher education” after they leave the bone marrow in order to mature. • T cell maturation occurs in the thymus. • Maturing T cells are called thymocytes. • Maturing T cells undergo positive and negative selection.

  11. Key Feature of the Thymus • Thymic stromal cells express the protein Notch. • In the absence of notch, no T cell maturation occurs. • If hematopoietic stem cells are transfected to express notch, then only T cells develop. • Thus, notch is a key to the differentiation of the immature T cells to mature T cells.

  12. T Cell Maturation in the Thymus • The T cell precursors enter the thymus as a double negative (CD4-CD8- or DN) cell. • They begin a process of dividing and differentiating that takes about 3 weeks. • Pass through 4 stages of DN cells • Become double positive (DP) cells • Ultimately become CD4+ or CD8+. • Leave the thymus as mature T cells.

  13. T Cell Maturation Strategic Steps • Move from BM to thymus and express… • CD44 for thymus localization • c-Kit for replication • CD25 (IL-2R) for IL-2-driven replication • TCR Rearrangements •  chain is first •  chain is second • Surface molecules • CD4-CD8- initially • CD4+CD8+ after  TCR • CD4+CD8- or CD4-CD8+

  14. T Cell Selection in the Thymus • Positive Selection = MHC Restriction: • Permits survival of only those T cells that recognize self-MHC molecules. • Thus, it is responsible for the selection of the self-MHC-restricted T cell repertoire. • Negative Selection = Self-Tolerance: • Eliminates T cells that respond too strongly to self MHC or with self MHC plus self peptides. • It is responsible for the development of a primary T cell repertoire that is self-tolerant. This process is called the development of Central Tolerance.

  15. Cost of T Cell Maturation • It is estimated that 98% of thymocytes do not mature into mature T cells. • Most are eliminated by apoptosis because • They fail to make productive TCR rearrangements • They fail to survive thymic selection

  16. T Cell Activation

  17. Multiple Genes Are Activated by Ag Binding • Immediate Early Genes • Expressed within 30 minutes of antigen recognition • Encode a number of transcription factors: c-Fos, c-Myc, c-Jun, NFAT, and NF-B • Early Genes • Expressed within 1-2 hrs of antigen recognition • Encode IL-2, IL-2R, IL-3, IL-6, IFN-, other proteins • Late Genes • Expressed more than 2 days after antigen recognition • Encode a number of adhesion molecules

  18. P56lck phosphorylates ITAMs on  chains, creating a docking site for ZAP-70. • ZAP-70 phosphorylates adaptor molecules that activate other enzymes. • Phospholipase C • activation causes • breakdown of • phosphoinositol • bisphosphate (PIP2) to • inositol 1,4,5 triphosphate • (IP3) • diacylglycerol (DAG)

  19. Inositol 1,4,5 triphosphate (IP3) • Causes rapid release of Ca++ • from endoplasmic reticulum • Opens Ca++ channels in the • cell membrane • Activates the transcription • factor NFAT that is required • for transcription of IL-2, IL-4 • Diacylglycerol (DAG) • Activates protein kinase C • which phosphorylates many • targets • Activates the transcription • factor NF-B that is in turn, • required for transcription of • IL-2 • Guanine nucleotide exchange factor (GEF) induces Ras and Rac pathways that lead to cell division.

  20. The RAS/MAP Kinase • Pathway • Ras is a small G protein • that, when activated by • GTP, initiates a cascade • of protein kinases called • the mitogen activated • protein kinase pathway • (MAP kinasepathway). • Activation of transcription complex of Fos/Jun/AP-1 that activates a number of genes, including those involved in initiation of cell division.

  21. Sensitivity of TCR:Antigen Binding for T Cell Activation • Binding of one TCR on a T cell to its cognate antigen is sufficient to trigger the activation of the T cell. • Incremental T cell activation occurs with more TCR:antigen bindings. • Maximal T cell activation occurs when 10 TCR:antigen bindings have occurred.

  22. Costimulatory Signals • Helper T cell activation requires two binding signals. • Signal 1: the initial signal generated by TCR:antigen recognition • Signal 2: the second signal (non-specific for antigen) is provided when CD28 on the T cell interacts with B7 on the antigen-presenting cell. • Helper T cell activation requires cytokine signals.

  23. Costimulatory Signal Regulation • Resting T cell • Expresses CD28 • Activation signal is transduced when CD28 is bound by B7 • CTLA-4 induced • Activated T cell • Expresses CTLA-4 in addition to CD28 • Inhibitory signal is transduced when CTLA-4 is bound by B7, providing a brake on activation and proliferation

  24. Clonal Anergy • What if there is no costimulatory signal mediated by B7 binding to CD28 • The T cell is in a non-responsive state (clonal anergy). • It cannot respond to the TCR:antigen binding signal.

  25. Superantigens • Some antigens can bind both to the MHC and to certain TCR molecules, without residing in the antigen groove of the MHC molecule. • Initiate a non-specific interaction • Stimulate many T cells of different antigenic specificities to divide and differentiate • Called superantigens • Activation of so many T cell clones can have serious consequences, such as the over induction of IFN- and TNF- associated with toxic shock.

  26. T Cell Differentiation

  27. The Naïve T Cell Population • T cells leave the thymus as naïve T cells. • There are about 2X as many CD4+ T cells as CD8+ T cells in the periphery. • The T cells are in G0, or the resting phase of the cell cycle. • The naïve T cells constantly circulate from blood, to lymph, to lymphoid tissues, and back to blood in a cycle that takes about 12-24 hrs. • If a naïve T cell encounters its cognate antigen in the lymph node, it remains there. • The rapid recirculation of naïve T cells is necessary because only about 1:105 naïve T cells has specificity for any given antigen.

  28. Th Cell Differentiation • Binding of the TCR to its cognate antigen initiates the primary response. • After about 24 hrs, the responding T cell enlarges to form a blast cell and begins to undergo rounds of cell division. • IL-2 synthesis is increased by 100-fold by induction of IL-2 mRNA synthesis and by stabilization of IL-2 mRNA. • IL-2 binding to the high affinity IL-2 receptor (also induced after antigen binding) activates the proliferation: 2-3 division/day for 4-5 days to generate a clone of responding T cells. • Some of the responding T cells become effector T cells; others become memory T cells.

  29. T Cell Apoptosis • After undergoing rapid proliferation, effector T cells must undergo apoptosis or we would become blobs of T cells. • FasL-mediated apoptosis: • Death in 2-4 hours • MHC/Ag-mediated apoptosis: • Death in 8-10 hours • Note that memory T cells do not undergo apoptosis.

  30. Effector T Cells • Effector T cells can be induced from naïve T cells or from memory T cells upon exposure to cognate antigen. • Effector T cells are short-lived, surviving for a few days to a few weeks. • Effector T cells can be of several types. • CD4+ Helper T cells • Th1 subset secretes IL-2, IFN-, TNF- and stimulates cell mediated immunity. • Th2 subset secretes IL-4, IL-5, IL-6, IL-10 and stimulates humoral (antibody mediated) immunity • CD8+ Cytotoxic T cells

  31. Memory T Cells • Memory T cells can be induced from naïve T cells or from effector T cells after antigenic activation and differentiation. • Memory T cells are long-lived, surviving for many years. • Memory T cells can be reactivated by re-exposure to cognate antigen to become effector cells (secondary response). • There are no identifying surface markers that can be used to differentiate memory and effector T cells. • While naïve T cells are almost exclusively activated by dendritic cells, memory T cells can be activated by macrophages, dendritic cells, and B cells (thought to be a function of high levels of adhesion molecules).

  32. Regulatory T Cells • The CD4+CD25+FoxP3+ subpopulation of T cells can suppress the immune response (regulatory T cells, Treg cells). • Others cells may also have regulatory activity. • Loss of Treg cells by Ab depletion has caused development of autoimmunity.

  33. James Cormack Because of the lack of T cell responsiveness to Candida albicans and low IgM levels, the physician suspects a T cell disorder. The physician knows (from checking uptodate.com) that among the potential causes of thrombocytemia, Wiskott-Aldrich syndrome stands out as being the only one that is an immunodeficiency condition. The physician contacts the Immune Deficiency Foundation and sends sample for genetic testing for suspected Wiskott-Aldrich syndrome. The results of the genetic testing confirm that James has Wiskott-Aldrich syndrome.

  34. James Cormack Patients with Wiscott-Aldrich syndrome have a mutation in the Wiscott-Aldrich protein (WASp). Individuals with mutant WASp have defects in IL-2 production, resulting in impaired T cell proliferation. Thus, individuals with mutant WASp protein is have impaired helper T cell induction of antibody production and impaired helper T cell induction of CD8+ T cell function. WASp is also required for platelet production. In the absence of platelets, bruising easily occurs The physician arranges for James to have a bone marrow transplant.

  35. Details of Thymic Maturation Events

  36. Stages of T Cell Maturation • DN1 cells (c-kit+, CD44high, CD25- cells) enter the thymus. • CD44high is needed for localization to thymus. • c-Kit+ is a receptor for stem cell factor and is needed for initiation of growth in the thymic environment. • DN1 cells respond to the thymic environment by beginning to proliferate and to express CD25 (IL-2R). • DN1 cells are capable of giving rise to all subsets of T cells.

  37. Stages of T Cell Maturation • DN2 cells have turned on synthesis of CD25 (c-kit+, CD44low, CD25+ cells). • They turn on RAG-1 and RAG-2 and begin rearranging TCR , , and . • TCR  does not begin rearrangement because its DNA region is too condensed. • Cells destined to express TCR  diverge from the other T cells with the transition from DN2 to DN3 and leave the thymus.

  38. Stages of T Cell Maturation • DN3 cells have turned off c-kit and CD44 (c-kit-, CD44-, CD25+ cells). • The DN3 cells halt their proliferation. • TCR  is rearranged. • It combines with a 33 kDa protein known as the pre-T chain. • This dimer associates with the CD3 group of molecules to form a complex called the pre-T cell receptor or the pre-TCR.

  39. T Cells Bearing the Pre-TCR • Once the Pre-TCR is produced, an activation signal can be transduced across the membrane to initiate several actions. • Indicates that the cell has made a TCR  chain and signals further proliferation and maturation. • Suppresses further rearrangement of TCR , resulting in allelic exclusion. • Permits the cell to rearrange the TCR  chain. • Induces developmental progression to the CD4+CD8+ double-positive T cell. • Delayed synthesis of TCR  chain gives a tremendous increase in the diversity of the T cells, since each T cell with a given  chain can express a different  chain.

  40. Stages of T Cell Maturation • DN4 cells turn off expression of CD25 (c-kit-, CD44-, CD25- cells). • Expression of CD4 and CD8 is turned on. • Double positive T cells (CD4+CD8+ T cells) • Rapid proliferation occurs, creating a clone of cells with the same TCR  chain. • After a period of time, proliferation stops and the TCR  chain is sythesized. • Delayed synthesis of TCR  chain gives a tremendous increase in the diversity of the T cells, since each T cell with a given  chain can express a different  chain. • Expression of a functional TCR permits the T cell to undergo positive and negative selection.

  41. Stages of T Cell Maturation • Double positive T cells (CD4+CD8+ T cells) lose one of their T cell markers and become CD4+ or CD8+ T cells. • The single-positive T cells undergo additional negative selection.

  42. Key Players in Activation • The RAS/MAP Kinase Pathway • Ras is a small G protein that, when activated by GTP, initiates a cascade of protein kinases called the mitogen-activated protein kinase pathway (MAP kinasepathway). • Activation of transcription complex of Fos/Jun/AP-1 that activates a number of genes, including those involved in initiation of cell division.

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