T-Cell Maturation, Activation, And Differentiation

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. T Cell Maturation

4. 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.

5. 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.

6. 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.

7. 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+

8. 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.

9. 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

10. T Cell Activation

11. 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

12. 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)

13. 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.

14. 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.

15. 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.

16. 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.

17. 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

18. 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.

19. 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.

20. T Cell Differentiation

21. 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.

22. 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.

23. 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.

24. 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

25. 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).

26. 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.

27. Details of Thymic Maturation Events

28. 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.

29. 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.

30. 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.

31. 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.

32. 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.

33. 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.