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. 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 1.Move from BM to thymus and express… a.CD44 for thymus localization b.c-Kit for replication c.CD25 (IL-2R) for IL-2- driven replication 2.TCR Rearrangements  chain is first  chain is second 3.Surface molecules a.CD4-CD8- initially b.CD4+CD8+ after  TCR c.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. P56 lck 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 (PIP 2 ) to inositol 1,4,5 triphosphate (IP 3 ) diacylglycerol (DAG)

13. Inositol 1,4,5 triphosphate (IP 3 ) –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 kinase pathway). – 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 G 0, 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 1:10 5 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 +, CD44 high, CD25 - cells) enter the thymus. –CD44 high 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 +, CD44 low, 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.